Ubiquitin conjugation assays

ABSTRACT

The invention relates to assays for measuring ubiquitin ligase activity and for identifying modulators of ubiquitin ligase enzymes.

This application is a continuation of application Ser. No. 09/826,312,filed Apr. 3, 2001 now U.S. Pat. No. 6,737,244, which is acontinuation-in part of application Ser. No. 09/542,497, filed on Apr.3, 2000 now U.S. Pat. No. 6,740 ,495; both of which are hereinincorporated by reference for all purposes.

FIELD OF THE INVENTION

This invention is directed to assays for measuring the activity ofubitquitination enzymes. The invention is also directed to assays foridentifying modulators of ubiquitination.

BACKGROUND OF THE INVENTION

Ubiquitin is a highly conserved 76 amino acid protein expressed in alleukaryotic cells. The levels of many intracellular proteins areregulated by a ubiquitin-dependent proteolytic process. This processinvolves the covalent ligation of ubiquitin to a target protein,resulting in a poly-ubiquitinated target protein which is rapidlydetected and degraded by the 26S proteasome.

The ubiquitination of these proteins is mediated by a cascade ofenzymatic activity. Ubiquitin is first activated in an ATP-dependentmanner by a ubiquitin activating enzyme (E1). The C-terminus of aubiquitin forms a high energy thiolester bond with E1. The ubiquitin isthen passed to a ubiquitin conjugating enzyme (E2; also called ubiquitincarrier protein), also linked to this second enzyme via a thiolesterbond. The ubiquitin is finally linked to its target protein to form aterminal isopeptide bond under the guidance of a ubiquitin ligase (E3).In this process, chains of ubiquitin are formed on the target protein,each covalently ligated to the next through the activity of E3.

The components of the ubiquitin ligation cascade have receivedconsiderable attention (for a review, see Weissman, Nature Reviews2:169–178 (2001)). E1 and E2 are structurally related and wellcharacterized enzymes. There are several species of E2 (at least 25 inmammals), some of which act in preferred pairs with specific E3 enzymesto confer specificity for different target proteins. While thenomenclature for E2 is not standardized across species, investigators inthe field have addressed this issue and the skilled artisan can readilyidentify various E2 proteins, as well as species homologues (See Haasand Siepmann, FASEB J. 11:1257–1268 (1997)).

E3 enzymes contain two separate activities: a ubiquitin ligase activityto conjugate ubiquitin to substrates and form polyubiquitin chains viaisopeptide bonds, and a targeting activity to physically bring theligase and substrate together. Substrate specificity of different E3enzymes is the major determinant in the selectivity of theubiquitin-dependent protein degradation process.

Some E3 ubiquitin ligases are known to have a single subunit responsiblefor the ligase activity. Such E3 ligases that have been characterizedinclude the HECT (homologous to E6-AP carboxy terminus) domain proteins,represented by the mammalian E6AP-E6 complex which functions as aubiquitin ligase for the tumor suppressor p53 and which is activated bypapillomavirus in cervical cancer (Huang et al., Science 286:1321–26(1999)). Single subunit ubiquitin ligases having a RING domain includeMdm2, which has also been shown to act as a ubiquitin ligase for p53, aswell as Mdm2 itself. Other RING domain, single subunit E3 ligasesinclude: TRAF6, involved in IKK activation; Cbl, which targets insulinand EGF; Sina/Siah, which targets DCC; Itchy, which is involved inhaematopoesis (B, T and mast cells); and IAP, involved with inhibitorsof apoptosis.

The best characterized E3 ligase is the APC (anaphase promotingcomplex), which is a multi-subunit complex that is required for bothentry into anaphase as well as exit from mitosis (see King et al.,Science 274:1652–59 (1996) for review). The APC plays a crucial role inregulating the passage of cells through anaphase by promotingubiquitin-dependent proteolysis of many proteins. In addition todegrading the mitotic B-type cyclin for inactivation of CDC2 kinaseactivity, the APC is also required for degradation of other proteins forsister chromatid separation and spindle disassambly. Most proteins knownto be degraded by the APC contain a conserved nine amino acid motifknown as the “destruction box” that targets them for ubiquitination andsubsequent degradation. However, proteins that are degraded during G1,including G1 cyclins, CDK inhibitors, transcription factors andsignaling intermediates, do not contain this conserved amino acid motif.Instead substrate phosphorylation appears to play an important role intargeting their interaction with an E3 ligase for ubiquitination (seeHershko et al., Ann. Rev. Biochem. 67:429–75 (1998)).

In eukaryotes, a family of complexes with E3 ligase activity play animportant role in regulating G1 progression. These complexes, calledSCF's, consist of at least three subunits, SKP1, Cullins (having atleast seven family members) and an F-box protein (of which hundreds ofspecies are known) which bind directly to and recruit the substrate tothe E3 complex. The combinatorial interactions between the SCF's and arecently discovered family of RING finger proteins, the ROC/APC 11proteins, have been shown to be the key elements conferring ligaseactivity to E3 protein complexes. Particular ROC/Cullin combinations canregulate specific cellular pathways, as exemplified by the function ofAPC11-APC2, involved in the proteolytic control of sister chromatidseparation and exit from telophase into G1 in mitosis (see King et al.,supra; Koepp et al., Cell 97:431–34 (1999)), and ROC1-Cullin 1, involvedin the proteolytic degradation of I_(κ)B_(α) in NF-_(κ)-B/I_(κ)Bmediated transcription regulation (Tan et al., Mol. Cell 3(4):527–533(1999); Laney et al., Cell 97:427–30 (1999)).

Because the E3 complex is the major determinant of selection for proteindegradation by the ubiquitin-dependent proteolytic process, modulatorsof E3 ligase activity may be used to upregulate or downregulate specificmolecules involved in cellular signal transduction. Disease processescan be treated by such up- or down regulation of signal transducers toenhance or dampen specific cellular responses. This principle has beenused in the design of a number of therapeutics, includingPhosphodiesterase inhibitors for airway disease and vascularinsufficiency, Kinase inhibitors for malignant transformation andProteasome inhibitors for inflammatory conditions such as arthritis.

Due to the importance of ubiquitination in cellular regulation and thewide array of different possible components in ubiquitin-dependentproteolysis, there is a need for a fast and simple means for assaying E3ligase activity. Furthermore, such an assay would be very useful for theidentification of modulators of E3 ligase. Accordingly, it is an objectof the present invention to provide methods of assaying ubiquitin ligaseactivity, which methods may further be used to identify modulators ofubiquitin ligase activity.

DESCRIPTION OF THE RELATED ART

Tan et al., supra, disclose that ROC1/Cul 1 catalyzes ubiquitinpolymerization in the absence of target protein substrate. Ohta et al.,Mol. Cell 3(4):535–541 (1999) disclose that APC11/APC2 also catalyzeubiquitin polymerization in the absence of target protein substrate, andthat this activity is dependent on the inclusion of the proper E2species. Rolfe et al., U.S. Pat. No. 5,968,761 disclose an assay foridentifying inhibitors of ubiquitination of a target regulatory protein.

SUMMARY OF THE INVENTION

The present invention provides methods for assaying ubiquitin ligaseactivity and screening for agents which modulate ubiquitin ligaseactivity. In one aspect, a method of assaying ubiquitin ligase activityis provided involving the steps of combining ubiquitin, E1, E2 and E3and measuring the amount of ubiquitin bound to E3. This method mayfurther involve combining a candidate ubiquitin ligase modulator in thecombining step. This method does not require a specific target proteinto be ubiquitinated. In a preferred embodiment, a substrate protein forubiquitination other than ubiquitin itself is specifically excluded.

In one embodiment of the assay described above, ubiquitin is in the formof tag1-ubiquitin. In another embodiment, E3 is in the form of tag2-E3.In these embodiments tag1 may be a label or a partner of a binding pair.In one embodiment, tag1 is a fluorescent label, in which case measuringthe amount of ubiquitin bound to E3 may be by measuring luminescence.

In another embodiment, tag1 is a member of a binding pair chosen fromthe group antigen, biotin and CBP. In this latter embodiment, thepartner of a binding pair may be labeled by indirect labeling, which maybe by a fluorescent label or a label enzyme. The label enzyme may behorseradish peroxidase, alkaline phosphatase or glucose oxidase. Whenthe indirect labeling is by a fluorescent label, measuring the amount ofubiquitin bound to E3 may be by measuring luminescence. In the case thatthe indirect labeling is by a label enzyme, said enzyme may be reactedwith a substrate which produces a fluorescent product, in which case,measuring the amount of ubiquitin bound to E3 may be by measuringluminescence. In one embodiment of the method above, tag1 is a FLAGantigen. In this embodiment, indirect labeling may be by anti-FLAG.

In one aspect of the above method, tag2 is a surface binding molecule,which may be His-tag. In this latter case, the assaying may be performedin a multi-well plate comprising a surface substrate comprising nickel.

In a different embodiment of the method above, when tag1 is afluorescent label, the combining step further includes combiningtag3-ubiquitin. Tag3 may be the second member of a FRET pair with tag1or it may be a quencher of tag1. In this embodiment, measuring theamount of ubiquitin bound to E3 may be by measuring fluorescentemission, which may involve measuring the fluorescent emission spectrum.In this last embodiment, the method may further comprise comparing themeasured fluorescent emission spectrum with the fluorescent emissionspectrum of unbound tag1- and tag3-ubiquitin. When measuring the amountof ubiquitin bound to E3 is by measuring the fluorescent emissionspectrum, this measuring may be continuous or at specific time pointsfollowing the original combining of materials.

In another aspect of the invention, a method of identifying modulatorsof ubiquitination enzymes is provided. This method involves combiningtag1-ubiquitin, a candidate modulator, E1, E2 and tag2-E3 and measuringthe amount of tag1-ubiquitin bound to tag2-E3. In another embodiment,this method further comprises combining tag1-ubiquitin, a candidatemodulator, E1 and tag2-E2 and measuring the amount of tag1-ubiquitinbound to the tag2-E2. In a preferred embodiment, target protein (i.e., asubstrate protein other than ubiquitin itself) is specifically excludedin the method.

In the embodiments of the method of identifying modulators ofubiquitination enzymes, tag1 may be a label or a partner of a bindingpair. If tag1 is a label, it may be a fluorescent label, in which case,measuring the amount of bound tag1-ubiquitin may be by measuringluminescence. If tag1 is a partner of a binding pair, the potentialbinding pair partners, labeling options and subsequent measuring optionsare substantially as described for tag1 above for the method of assayingubiquitin ligase activity.

In the above method of identifying modulators of ubiquitination enzymes,tag2 and tag3 may be surface substrate binding molecules. Options forsuch molecules and conditions for performing the method are as describedfor the method of assaying ubiquitin ligase activity.

In another aspect of the invention, a method of assaying ubiquitinationenzyme activity is provided. This method comprises combiningtag1-ubiquitin and tag2-ubiquitin, E1, E2 and E3 under conditions inwhich ubiquitination can take place and measuring the amount or rate ofubiquitination. In this embodiment, tag1 and tag2 constitute a FRET pairor tag1 is a fluorescent label and tag2 is a quencher of tag1. In oneembodiment, the method includes combining a candidate ubiquitinationmodulator with the other components. In a preferred embodiment of thismethod, measuring is by measuring the fluorescent emission spectrum fromthe combination, preferably continuously or at specific time pointsfollowing combining the components. These measurements may be comparedto the fluorescent emission spectrum of unbound tag1 and tag2 ubiquitin.

Also provided herein is a method of identifying a ubiquitinationmodulator. This method involved combining a candidate ubiquitinationmodulator, tag1-ubiquitin and tag2-ubiquitin, E1, E2 and E3 underconditions in which ubiquitination can take place and measuring theamount or rate of ubiquitination. In this embodiment, tag1 and tag2constitute a FRET pair or tag1 is a fluorescent label and tag2 is aquencher of tag1. In one embodiment, the method includes combining acandidate ubiquitination modulator with the other components. In apreferred embodiment of this method, measuring is by measuring thefluorescent emission spectrum from the combination, preferablycontinuously or at specific time points following combining thecomponents. These measurements may be compared to the fluorescentemission spectrum of unbound tag1 and tag2 ubiquitin.

In the latter two assays described, the ubiquitin may be in the formtag1,3-ubiquitin and tag2,3-ubiquitin, wherein tag3 is a member of abinding pair, preferably FLAG. In another embodiment of these assays, E3may be in the form of tag4-E3, wherein tag4 is a surface substratebonding molecule.

In still another aspect of the invention, compositions are provided foruse in assaying ubiquitination. The composition comprises tag1-ubiquitinand tag2-ubiquitin, wherein tag1 and tag2 constitute a FRET pair or tag1is a fluorescent label and tag2 is a quencher of tag1. In oneembodiment, the composition further comprises E1, E2 and E3. In apreferred embodiment, the composition further comprises a candidateubiquitination modulator. In yet another embodiment, the compositioncomprises a target protein.

In addition, provided herein are compositions for use in assaying for aubiquitination modulator. The composition comprises a candidateubiquitination modulator, tag1-ubiquitin and tag2-ubiquitin, whereintag1 and tag2 constitute a FRET pair or tag1 is a fluorescent label andtag2 is a quencher of tag1. In one embodiment, the composition furthercomprises E1, E2 and E3. In one embodiment, the composition comprises atarget protein.

In preferred embodiments of the assays and compositions described above,E2 is selected from the group consisting of Ubc5, Ubc3, Ubc4 and UbcX.In ap referred embodiment, E3 comprises a RING finger protein,preferably selected from the group consisting of ROC1, ROC2 and APC11.In a preferred embodiment, E3 comprises a Cullin, preferably selectedfrom the group consisting of CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5 andAPC2. In a preferred embodiment, E3 comprises a RING fingerprotein/Cullin combination, preferably selected from the groupconsisting of APC11/APC2, ROC1/CUL1, ROC1/CUL2 and ROC2/CUL5.

Other aspects of the invention will become apparent to the skilledartisan from the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative amounts of fluorescently labeled ubiquitin ina ubiquitin activating and conjugating assay. In these experiments, E2is His-Ubch5c.

FIG. 2 shows the relative amounts of ubiquitin ligase activity resultingfrom various combinations of ubiquitination enzymes. In theseexperiments, E3 comprises the RING finger protein ROC1 and the CullinCul1.

FIG. 3 shows relative ubiquitin ligase activity in an assay combiningubiquitin, E1, E2 and E3. FIG. 3A shows relative ubiquitin ligaseactivity using varying amounts of E1 in the presence and absence ofDMSO. FIG. 3B shows relative ubiquitin ligase activity using varyingamounts of ubiquitin and E3.

FIG. 4 shows the signal to noise ratio of fluorescent label in aubiquitin ligase activity assay utilizing FLAG-ubiquitin (SEQ ID NO:14)and an anti-FLAG (DYKDDDDK; SEQ ID NO:16)/anti-mouse antibody conjugatedto HRP and Luminol fluorescent HRP substrate. The signal was measuredfrom a reaction composition comprising E1, E2 and E3, which E3specifically bound the reaction receptacle surface substrate. Thebackground was measured as the amount of fluorescence present afterperforming the assay in the absence of E3.

FIG. 5 shows the concentration-dependent effect of two ubiquitin ligaseactivity modulators in assays measuring ubiquitin ligase activity withtwo different E3 enzymes. FIG. 5A shows a concentration-dependentreduction in ubiquitin ligase activity in assays comprising eitherROC1/Cul1 or ROC2/Cul5 as the components of the E3 ubiquitin ligase.FIG. 5B shows a slightly different pattern of concentration-dependentreduction of ubiquitin ligase activity for another modulator.

FIG. 6 shows the proportions of ubiquitin ligase activity and ubiquitinconjugating activity in the presence and absence of two candidateubiquitin ligase enzyme modulators for combinations of E1, E2 and E3 andcombinations of enzymes E1 and E2. FIG. 6A shows a candidateubiquitination enzyme modulator that affects only E3. FIG. 6B showscandidate ubiquitination enzyme modulator that affects enzymes otherthan E3.

FIG. 7 shows the concentration-dependent effects of two candidateubiquitin ligase modulators on ubiquitin ligase activity and ubiquitinconjugating activity. FIG. 7A shows the results for a candidatemodulator having a concentration-dependent effect on ubiquitin ligaseactivity (E1+E2+E3), but not have on ubiquitin conjugating activity(E1+E2), thus affecting only the E3 ligase. FIG. 7B shows the resultsfor a candidate modulator having a concentration-dependent effect onboth ubiquitin conjugating activity and ubiquitin ligase activity, thusaffecting a component other than the E3 ligase.

FIGS. 8A and 8B show the nucleic acid sequence encoding rabbit E1 andthe amino acid sequence of rabbit E1, respectively.

FIGS. 9A and 9B show the nucleic acid sequence encoding the E2 Ubc5c andthe amino acid sequence of the E2 Ubc5c, respectively.

FIG. 10 shows the amino acid sequence of the RING finger protein APC11.

FIG. 11 shows the amino acid sequence of the RING finger protein ROC1.

FIGS. 12A and 12B show the nucleic acid sequence encoding the RINGfinger protein ROC2 and the amino acid sequence of ROC2, respectively.

FIGS. 13A and 13B show the nucleic acid sequence encoding the CullinCUL5 and the amino acid sequence of CUL5, respectively.

FIGS. 14A and 14B show the nucleic acid sequence encoding the CullinAPC2 and the amino acid sequence of APC2, respectively.

FIGS. 15A, 15B and 15C (SEQ ID NOS:13–15) show the amino acid sequencesof human ubiquitin, FLAG-ubiquitin and FLAG-Cys-ubiquitin, respectively.The FLAG (DYKDDDDK; SEQ ID NO:16) and FLAG-Cys (SEQ ID NO:17) portionsof the sequence are shown in bold.

FIGS. 16A and 16B show the E3 ligase dependent incorporation ofFLAG-Ala-Cys-ubiquitin (SEQ ID NO:18) labeled with FRET fluorophoresinto E3-ubiquitin complex. Isolation by HPLC shows emissions from freeubiquitin and ubiquitin attached to the E3 ligase. The traces showfluorescent emission at the wavelength described below, under excitationat 336 nm, the optimal excitation wavelength for IAEDANS. FIG. 16A showsthe fluorescence signals of IAEDANS (490 nm; larger peak) andfluorescein (515 nm; smaller peak) labeled ubiquitin followingcombination with E1 and E2 only. The free ubiquitin was isolated usinghigh performance liquid chromatography (HPLC). FIG. 16B shows thefluorescence signals of IAEDANS (490 nm; larger peak at each elutionvolume) and fluorescein (515 nm; smaller peak at each elution volume)labeled ubiquitin following combination with E1 and E2 and E3 (Roc l/Cul1). The dashed line shows optical density of the protein solution(scale on right), revealing the high sensitivity of the fluorophoresdespite a very low concentration of protein.

FIG. 17 shows the fluorescence emission spectra of free ubiquitinlabeled with the FRET donor/acceptor pair EDANS and fluorescein underexcitation at 336 nm. The dashed line shows the emission spectra of freelabeled ubiquitin (reactants), while the solid line shows the emissionspectra of labeled ubiquitin bound to E3 (products). The greatlyincreased 515:490 nm emission ratio of the E3-bound ubiquitin ascompared with the free ubiquitin shows the energy transfer from theEDANS donor to the fluorescein acceptor of this FRET donor/acceptorpair.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for assaying ubiquitinligase activity. In a broad embodiment, the method provides measuringubiquitin ligase activity directly where the reaction has occurred, thusobviating the need for target proteins and subsequent analysis such asseparating ligated from unligated material in an SDS PAGE procedure.This allows multi-well array analysis and high throughput screeningtechniques for modulators of ubiquitination activity. In addition, thepresent methods allow the analysis of many different combinations of E3components and E2/E3 combinations, without requiring prioridentification of specific target substrates.

In general, the method involves combining ubiquitin and ubiquitinligation enzymes and measuring the amount of ubiquitin ligated to aubiquitination substrate protein. In a preferred embodiment, theubiquitination substrate protein is ubiquitin itself, and what ismeasured is poly-ubiquitin chains produced in the ligase reaction.Therefore, as used herein, “ubiquitination substrate protein” means aprotein to which ubiquitin is bound through the activity ofubiquitination enzymes and “ubiquitination” and grammatical equivalentsthereof means the binding of ubiquitin to a substrate protein.

In a preferred embodiment, no specific target protein is used to measureubiquitin ligase activity. By “target protein” herein is meant a proteinother than ubiquitin to which ubiquitin is ligated by ubiquitinationenzymes. In this embodiment, preferably, the poly-ubiquitin chainsmeasured are bound to E3. In another preferred embodiment, thepoly-ubiquitin chains measured may be bound to E3 or not.

In a preferred embodiment, E3 is attached to the surface of a reactionvessel, such as the well of a multi-well plate. This embodimentfacilitates the separation of ligated ubiquitin from unligatedubiquitin. Means of attaching E3 to the surface of a reaction vessel aredescribed below. This embodiment allows the ubiquitin ligase reactionand detection and measurement of ligated ubiquitin to occur in the samevessel, making the assay useful for high-throughput screeningapplications.

In another preferred embodiment, E3 is free in solution. In thisembodiment, ubiquitination activity is monitored using a system thatproduces a signal which varies with the extent of ubiquitination, suchas the fluorescence resonance energy transfer (FRET) system described indetail below.

In a preferred embodiment, the ubiquitin is labeled, either directly orindirectly, as further described below, and the amount of label ismeasured. This allows for easy and rapid detection and measurement ofligated ubiquitin, making the assay useful for high-throughput screeningapplications. In one preferred embodiment, the signal of the labelvaries with the extent of ubiquitination, such as in the FRET systemdescribed below. One of ordinary skill in the art will recognize theapplicability of the present invention to screening for agents whichmodulate ubiquitination.

Accordingly, the present invention provides methods and compositions forassaying ubiquitin ligase activity. By “ubiquitin” herein is meant apolypeptide which is ligated to another polypeptide by ubiquitin ligaseenzymes. The ubiquitin can be from any species of organism, preferably aeukaryotic species. Preferably, the ubiquitin is mammalian. Morepreferably, the ubiquitin is human ubiquitin. In a most preferredembodiment, the ubiquitin has the amino acid sequence depicted in FIG.15A.

In a preferred embodiment, when ubiquitin is ligated to a targetprotein, that protein is targeted for degradation by the 26S proteasome.

Preferred embodiments of the invention utilize a 76 amino acid humanubiquitin. Other embodiments utilize variants of ubiquitin, as furtherdescribed below.

Also encompassed by “ubiquitin” is naturally occurring alleles andman-made variants of such a 76 amino acid polypeptide. In a preferredembodiment, the ubiquitin has the amino acid sequence of that depictedin ATCC accession number P02248, incorporated herein by reference. ATCCaccession numbers are found in Genbank. Sequences of GenBank accessionnumbers are incorporated herein by reference. GenBank is known in theart, see, e.g., Benson, D A, et al., Nucleic Acids Research 26:1–7(1998) and http://www.ncbi.nlm.nih.gov/. Preferably, the ubiquitin hasthe amino acid sequence depicted in FIG. 15A. In a preferred embodiment,variants of ubiquitin have an overall amino acid sequence identity ofpreferably greater than about 75%, more preferably greater than about80%, even more preferably greater than about 85% and most preferablygreater than 90% of the amino acid sequence depicted in FIG. 15A. Insome embodiments the sequence identity will be as high as about 93 to 95or 98%.

In another preferred embodiment, a ubiquitin protein has an overallsequence similarity with the amino acid sequence depicted in FIG. 15A ofgreater than about 80%, more preferably greater than about 85%, evenmore preferably greater than about 90% and most preferably greater than93%. In some embodiments the sequence identity will be as high as about95 to 98 or 99%.

As is known in the art, a number of different programs can be used toidentify whether a protein (or nucleic acid as discussed below) hassequence identity or similarity to a known sequence. Sequence identityand/or similarity is determined using standard techniques known in theart, including, but not limited to, the local sequence identityalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thesequence identity alignment algorithm of Needleman & Wunsch, J. Mol.Biol. 48:443 (1970), by the search for similarity method of Pearson &Lipman, PNAS USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12:387–395 (1984), preferably using the defaultsettings, or by inspection. Preferably, percent identity is calculatedby FastDB based upon the following parameters: mismatch penalty of 1;gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30,“Current Methods in Sequence Comparison and Analysis,” MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp 127–149(1988), Alan R. Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351–360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151–153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403–410, (1990) and Karlin etal., PNAS USA 90:5873–5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul et al.,Methods in Enzymology, 266: 460–480 (1996);http://blast.wustl/edu/blast/README.html]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al. Nucleic Acids Res. 25:3389–3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to ˜22 bits.

A percent amino acid sequence identity value is determined by the numberof matching identical residues divided by the total number of residuesof the “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the amino acid sequence depictd in FIG. 15A, itis understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than that of the sequencedepicted in FIG. 15A, as discussed below, will be determined using thenumber of amino acids in the shorter sequence, in one embodiment. Inpercent identity calculations relative weight is not assigned to variousmanifestations of sequence variation, such as, insertions, deletions,substitutions, etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

Ubiquitin proteins of the present invention may be shorter or longerthan the amino acid sequence depicted in FIG. 15A. Thus, in a preferredembodiment, included within the definition of ubiquitin are portions orfragments of the amino acid sequence depicted in FIG. 15A. In oneembodiment herein, fragments of ubiquitin are considered ubiquitinproteins if they are ligated to another polypeptide by ubiquitin ligaseenzymes.

In addition, as is more fully outlined below, ubiquitin can be madelonger than the amino acid sequence depicted in FIG. 15A; for example,by the addition of tags, the addition of other fusion sequences, or theelucidation of additional coding and non-coding sequences. As describedbelow, the fusion of a ubiquitin peptide to a fluorescent peptide, suchas Green Fluorescent Peptide (GFP), is particularly preferred.

The ubiquitin protein, as well as other proteins of the presentinvention, are preferably recombinant. A “recombinant protein” is aprotein made using recombinant techniques, i.e. through the expressionof a recombinant nucleic acid as described below. In a preferredembodiment, the ubiquitin of the invention is made through theexpression of a nucleic acid as depicted in ATCC accession number M26880or AB003730, or a fragment thereof. In a most preferred embodiment, thenucleic acid encodes the amino acid sequence depicted in FIG. 15A. Arecombinant protein is distinguished from naturally occurring protein byat least one or more characteristics. For example, the protein may beisolated or purified away from some or all of the proteins and compoundswith which it is normally associated in its wild type host, and thus maybe substantially pure. For example, an isolated protein is unaccompaniedby at least some of the material with which it is normally associated inits natural state, preferably constituting at least about 0.5%, morepreferably at least about 5% by weight of the total protein in a givensample. A substantially pure protein comprises at least about 75% byweight of the total protein, with at least about 80% being preferred,and at least about 90% being particularly preferred. The definitionincludes the production of a protein from one organism in a differentorganism or host cell. Alternatively, the protein may be made at asignificantly higher concentration than is normally seen, through theuse of an inducible promoter or high expression promoter, such that theprotein is made at increased concentration levels. Alternatively, theprotein may be in a form not normally found in nature, as in theaddition of an epitope tag or amino acid substitutions, insertions anddeletions, as discussed below.

As used herein and further defined below, “nucleic acid” may refer toeither DNA or RNA, or molecules which contain both deoxy- andribonucleotides. The nucleic acids include genomic DNA, cDNA andoligonucleotides including sense and anti-sense nucleic acids. Suchnucleic acids may also contain modifications in the ribose-phosphatebackbone to increase stability and half life of such molecules inphysiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequences depicted in FIGS. 1 and 3 also include the complement ofthe sequence. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid by endonucleases, in a form not normallyfound in nature. Thus an isolated nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e. using the in vivo cellular machinery of the hostcell rather than in vitro manipulations; however, such nucleic acids,once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention.

The terms “polypeptide” and “protein” may be used interchangeablythroughout this application and mean at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradation.

In one embodiment, the present invention provides compositionscontaining protein variants, for example ubiquitin, E1, E2 and/or E3variants. These variants fall into one or more of three classes:substitutional, insertional or deletional variants. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding a protein of the present compositions, using cassetteor PCR mutagenesis or other techniques well known in the art, to produceDNA encoding the variant, and thereafter expressing the DNA inrecombinant cell culture as outlined above. However, variant proteinfragments having up to about 100–150 residues may be prepared by invitro synthesis using established techniques. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the protein amino acid sequence. The variantstypically exhibit the same qualitative biological activity as thenaturally occurring analogue, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed variants screened for theoptimal desired activity. Techniques for making substitution mutationsat predetermined sites in DNA having a known sequence are well known,for example, M13 primer mutagenesis and PCR mutagenesis. Rapidproduction of many variants may be done using techniques such as themethod of gene shuffling, whereby fragments of similar variants of anucleotide sequence are allowed to recombine to produce new variantcombinations. Examples of such techniques are found in U.S. Pat. Nos.5,605,703; 5,811,238; 5,873,458; 5,830,696; 5,939,250; 5,763,239;5,965,408; and 5,945,325, each of which is incorporated by referenceherein in its entirety. Screening of the mutants is done using ubiquitinligase activity assays of the present invention.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the protein are desired,substitutions are generally made in accordance with the following chart:

CHART I Original Exemplary Residue Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro His Asn, Gln IleLeu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, TyrSer Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the proteins as needed. Alternatively, the variantmay be designed such that the biological activity of the protein isaltered. For example, glycosylation sites may be altered or removed.

Covalent modifications of polypeptides are included within the scope ofthis invention. One type of covalent modification includes reactingtargeted amino acid residues of a polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N-or C-terminal residues of a polypeptide. Derivatization withbifunctional agents is useful, for instance, for crosslinking a proteinto a water-insoluble support matrix or surface for use in the method forscreening assays, as is more fully described below. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, -hydroxy-succinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidyl-propionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman &Co., San Francisco, pp. 79–86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of a polypeptide included withinthe scope of this invention comprises altering the native glycosylationpattern of the polypeptide. “Altering the native glycosylation pattern”is intended for purposes herein to mean deleting one or morecarbohydrate moieties found in native sequence polypeptide, and/oradding one or more glycosylation sites that are not present in thenative sequence polypeptide.

Addition of glycosylation sites to polypeptides may be accomplished byaltering the amino acid sequence thereof. The alteration may be made,for example, by the addition of, or substitution by, one or more serineor threonine residues to the native sequence polypeptide (for O-linkedglycosylation sites). The amino acid sequence may optionally be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on apolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Such methods are described in the art, e.g., in WO 87/05330published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259–306 (1981).

Removal of carbohydrate moieties present on the polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo-and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of a protein comprises linking thepolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

Polypeptides of the present invention may also be modified in a way toform chimeric molecules comprising a first polypeptide fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of a ubiquitin polypeptide (or anE2 or an E3, as defined below) with a tag polypeptide which provides anepitope to which an anti-tag antibody can selectively bind. The epitopetag is generally placed at the amino-or carboxyl-terminus of thepolypeptide. The presence of such epitope-tagged forms of a polypeptidecan be detected using an antibody against the tag polypeptide. Also,providing an epitope tag enables the polypeptide to be readily purifiedby affinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. In an alternativeembodiment, the chimeric molecule may comprise a fusion of a polypeptidedisclosed herein with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule, such afusion could be to the Fc region of an IgG molecule. Tags for componentsof the invention are defined and described in detail below.

The present invention provides methods for assaying ubiquitin ligaseactivity. By “ubiquitin ligase” is meant a ubiquitination enzyme capableof catalyzing the covalent binding of a ubiquitin to another protein.Preferred embodiments of the invention involve combining ubiquitin andubiquitination enzymes, including ubiquitin ligase, under conditions inwhich ubiquitination may take place, and measuring the amount ofubiquitin (poly-ubiquitin) bound to the ubiquitin ligase. In a preferredembodiment, the ubiquitin ligase is an E3 ubiquitin ligase, definedbelow.

Embodiments of the present invention involve binding ubiquitin to aubiquitination substrate protein. By “ubiquitination substrate protein”is meant a protein to which the ubiquitin ligase can catalyze thecovalent binding of ubiquitin and includes target proteins andubiquitin. In a preferred embodiment, the ubiquitination substrateprotein is ubiquitin and the ubiquitin ligase catalyzes the formation ofpolyubiquitin chains. In a preferred embodiment, the polyubiquitinchains are formed by the ubiquitin ligase in the absence of any targetprotein.

In one aspect, the invention provides methods for assayingubiquitination. In these assays, the interaction of the differentubiquitination enzymes, the interaction of different subunits ofindividual ubiquitination enzymes, and the influence of candidateubiquitination modulators can be observed and measured.

In a preferred embodiment, the invention is directed to a method ofassaying ubiquitin ligase activity. By “ubiquitin ligase activity”,“ubiquitin ligation” and grammatical equivalents thereof is meant thecatalysis of the covalent binding of ubiquitin to a substrate protein.Preferably, each ubiquitin is bound such that a subsequent ubiquitinpolypeptide may be bound to it, to form chains comprising a plurality ofubiquitin molecules. In a preferred embodiment, ubiquitin ligaseactivity occurs in the absence of target proteins, thus the substrateprotein is ubiquitin.

In a preferred embodiment, the invention is additionally directed to amethod of assaying ubiquitin activating activity. By “ubiquitinactivating activity”, “ubiquitin activation” and grammatical equivalentsthereof is meant the binding of ubiquitin and E1. Preferably, E1 forms ahigh energy thiolester bond with the ubiquitin.

In a preferred embodiment, the invention is also directed to a method ofassaying ubiquitin conjugating activity. By “ubiquitin conjugatingactivity”, “ubiquitin conjugation” and grammatical equivalents thereofis meant the binding of activated ubiquitin with an E2. As will beappreciated by those in the art, due to the presence of the high energythiolester bond in the E2-ubiquitin conjugate, conjugated ubiquitin maybe joined to other ubiquitin at a low rate in the absence of thecatalytic activity of E3. Therefore, some of the ubiquitin measured in aubiquitin conjugating activity assay will be in the form ofpoly-ubiquitin.

The present invention provides methods and compositions comprisingcombining ubiquitin with other components. By “combining” is meant theaddition of the various components into a receptacle under conditions inwhich ubiquitin ligase activity or ubiquitination may take place. In apreferred embodiment, the receptacle is a well of a 96 well plate orother commercially available multiwell plate. In an alternate preferredembodiment, the receptacle is the reaction vessel of a FACS machine.Other receptacles useful in the present invention include, but are notlimited to 384 well plates and 1536 well plates. Still other receptaclesuseful in the present invention will be apparent to the skilled artisan.

The addition of the components may be sequential or in a predeterminedorder or grouping, as long as the conditions amenable to ubiquitinligase activity are obtained. Such conditions are well known in the art,and further guidance is provided below.

In a preferred embodiment, one or more components of the presentinvention comprise a tag. By “tag” is meant an attached molecule ormolecules useful for the identification or isolation of the attachedcomponent. Components having a tag are referred to as “tag-X”, wherein Xis the component. For example, a ubiquitin comprising a tag is referredto herein as “tag-ubiquitin”. Preferably, the tag is covalently bound tothe attached component. When more than one component of a combinationhas a tag, the tags will be numbered for identification, for example“tag1-ubiquitin”. Components may comprise more than one tag, in whichcase each tag will be numbered, for example “tag1,2-ubiquitin”.Preferred tags include, but are not limited to, a label, a partner of abinding pair, and a surface substrate binding molecule. As will beevident to the skilled artisan, many molecules may find use as more thanone type of tag, depending upon how the tag is used.

By “label” is meant a molecule that can be directly (i.e., a primarylabel) or indirectly (i.e., a secondary label) detected; for example alabel can be visualized and/or measured or otherwise identified so thatits presence or absence can be known. As will be appreciated by those inthe art, the manner in which this is done will depend on the label.Preferred labels include, but are not limited to, fluorescent labels,label enzymes and radioisotopes.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 andOregon green. Suitable optical dyes are described in the 1996 MolecularProbes Handbook by Richard P. Haugland, hereby expressly incorporated byreference. Suitable fluorescent labels also include, but are not limitedto, green fluorescent protein (GFP; Chalfie, et al., Science263(5148):802–805 (Feb. 11, 1994); and EGFP; Clontech—Genbank AccessionNumber U55762), blue fluorescent protein (BFP; 1. QuantumBiotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor,Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques24(3):462–471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178–182(1996)), enhanced yellow fluorescent protein (EYFP; 1. ClontechLaboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303),luciferase (Ichiki, et al., J. Immunol. 150(12):5408–5417 (1993)),β-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603–2607(April 1988)) and Renilla WO 92/15673; WO 95/07463; WO 98/14605; WO98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S. Pat. No. 5,418,155;U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No.5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; U.S. Pat.No. 5,876,995; and U.S. Pat. No. 5,925,558) All of the above-citedreferences are expressly incorporated herein by reference.

In some instances, multiple fluorescent labels are employed. In apreferred embodiment, at least two fluorescent labels are used which aremembers of a fluorescence resonance energy transfer (FRET) pair. FRET isphenomenon known in the art wherein excitation of one fluorescent dye istransferred to another without emission of a photon. A FRET pairconsists of a donor fluorophore and an acceptor fluorophore. Thefluorescence emission spectrum of the donor and the fluorescenceabsorption spectrum of the acceptor must overlap, and the two moleculesmust be in close proximity. The distance between donor and acceptor atwhich 50% of donors are deactivated (transfer energy to the acceptor) isdefined by the Förster radius (R_(o)), which is typically 10–100 Å.Changes in the fluorescence emission spectrum comprising FRET pairs canbe detected, indicating changes in the number of that are in closeproximity (i.e., within 100 Å of each other). This will typically resultfrom the binding or dissociation of two molecules, one of which islabeled with a FRET donor and the other of which is labeled with a FRETacceptor, wherein such binding brings the FRET pair in close proximity.Binding of such molecules will result in an increased fluorescenceemission of the acceptor and/or quenching of the fluorescence emissionof the donor.

FRET pairs (donor/acceptor) useful in the invention include, but are notlimited to, EDANS/fluorescien, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy5, fluorescein/Cy 5.5 and fluorescein/LC Red 705.

In another aspect of FRET, a fluorescent donor molecule and anonfluorescent acceptor molecule (“quencher”) may be employed. In thisapplication, fluorescent emission of the donor will increase whenquencher is displaced from close proximity to the donor and fluorescentemission will decrease when the quencher is brought into close proximityto the donor. Useful quenchers include, but are not limited to, DABCYL,QSY 7 and QSY 33. Useful fluorescent donor/quencher pairs include, butare not limited to EDANS/DABCYL, Texas Red/DABCYL, BODIPY/DABCYL,Lucifer yellow/DABCYL, coumarin/DABCYL and fluorescein/QSY 7 dye.

The skilled artisan will appreciate that FRET and fluorescence quenchingallow for monitoring of binding of labeled molecules over time,providing continuous information regarding the time course of bindingreactions.

It is important to remember that ubiquitin is ligated to substrateprotein by its terminal carboxyl group to a lysine residue, includinglysine residues on other ubiquitin. Therefore, attachment of labels orother tags should not interfere with either of these active groups onthe ubiquitin. Amino acids may be added to the sequence of protein,through means well known in the art and described herein, for theexpress purpose of providing a point of attachment for a label. In apreferred embodiment, one or more amino acids are added to the sequenceof a component for attaching a tag thereto, preferably a fluorescentlabel. In a preferred embodiment, the amino acid to which a fluorescentlabel is attached is Cysteine.

By “label enzyme” is meant an enzyme which may be reacted in thepresence of a label enzyme substrate which produces a detectableproduct. Suitable label enzymes for use in the present invention includebut are not limited to, horseradish peroxidase, alkaline phosphatase andglucose oxidase. Methods for the use of such substrates are well knownin the art. The presence of the label enzyme is generally revealedthrough the enzyme's catalysis of a reaction with a label enzymesubstrate, producing an identifiable product. Such products may beopaque, such as the reaction of horseradish peroxidase with tetramethylbenzedine, and may have a variety of colors. Other label enzymesubstrates, such as Luminol (available from Pierce Chemical Co.), havebeen developed that produce fluorescent reaction products. Methods foridentifying label enzymes with label enzyme substrates are well known inthe art and many commercial kits are available. Examples and methods forthe use of various label enzymes are described in Savage et al.,Previews 247:6–9 (1998), Young, J. Virol. Methods 24:227–236 (1989),which are each hereby incorporated by reference in their entirety.

By “radioisotope” is meant any radioactive molecule. Suitableradioisotopes for use in the invention include, but are not limited to¹⁴C, ³H, ³²P, ³³P, ³⁵S, ¹²⁵I, and ¹³¹I. The use of radioisotopes aslabels is well known in the art.

In addition, labels may be indirectly detected, that is, the tag is apartner of a binding pair. By “partner of a binding pair” is meant oneof a first and a second moiety, wherein said first and said secondmoiety have a specific binding affinity for each other. Suitable bindingpairs for use in the invention include, but are not limited to,antigens/antibodies (for example, digoxigenin/anti-digoxigenin,dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl,Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, andrhodamine/anti-rhodamine), biotin/avidin (or biotin/streptavidin) andcalmodulin binding protein (CBP)/calmodulin. Other suitable bindingpairs include polypeptides such as the FLAG-peptide (DYKDDDDK; SEQ IDNO:16) [Hopp et al., BioTechnology, 6:1204–1210 (1988)]; the KT3 epitopepeptide [Martin et al., Science 255:192–194 (1992)]; tubulin epitopepeptide [Skinner et al., J. Biol. Chem 266:15163–15166 (1991)]; and theT7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.Acad. Sci. USA, 87:6393–6397 (1990)] and the antibodies each thereto.Generally, in a preferred embodiment, the smaller of the binding pairpartners serves as the tag, as steric considerations in ubiquitinligation may be important. As will be appreciated by those in the art,binding pair partners may be used in applications other than forlabeling, as is further described below.

As will be appreciated by those in the art, a partner of one bindingpair may also be a partner of another binding pair. For example, anantigen (first moiety) may bind to a first antibody (second moiety)which may, in turn, be an antigen for a second antibody (third moiety).It will be further appreciated that such a circumstance allows indirectbinding of a first moiety and a third moiety via an intermediary secondmoiety that is a binding pair partner to each.

As will be appreciated by those in the art, a partner of a binding pairmay comprise a label, as described above. It will further be appreciatedthat this allows for a tag to be indirectly labeled upon the binding ofa binding partner comprising a label. Attaching a label to a tag whichis a partner of a binding pair, as just described, is referred to hereinas “indirect labeling”.

By “surface substrate binding molecule” and grammatical equivalentsthereof is meant a molecule have binding affinity for a specific surfacesubstrate, which substrate is generally a member of a binding pairapplied, incorporated or otherwise attached to a surface. Suitablesurface substrate binding molecules and their surface substratesinclude, but are not limited to poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags and Nickel substrate; theGlutathione-S Transferase tag and its antibody substrate (available fromPierce Chemical); the flu HA tag polypeptide and its antibody 12CA5substrate [Field et al., Mol. Cell. Biol., 8:2159–2165 (1988)]; thec-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibody substratesthereto [Evan et al., Molecular and Cellular Biology, 5:3610–3616(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody substrate [Paborsky et al., Protein Engineering, 3(6):547–553(1990)]. In general, surface binding substrate molecules useful in thepresent invention include, but are not limited to, polyhistidinestructures (His-tags) that bind nickel substrates, antigens that bind tosurface substrates comprising antibody, haptens that bind to avidinsubstrate (e.g., biotin) and CBP that binds to surface substratecomprising calmodulin.

Production of antibody-embedded substrates is well known; see Slinkin etal., Bioconj. Chem. 2:342–348 (1991); Torchilin et al., supra;Trubetskoy et al., Bioconj. Chem. 3:323–327 (1992); King et al., CancerRes. 54:6176–6185 (1994); and Wilbur et al., Bioconjugate Chem.5:220–235 (1994) (all of which are hereby expressly incorporated byreference), and attachment of or production of proteins with antigens isdescribed above.

Calmodulin-embedded substrates are commercially available , andproduction of proteins with CBP is described in Simcox et al.,Strategies 8:40–43 (1995), which is hereby incorporated by reference inits entirety.

As will be appreciated by those in the art, tag-components of theinvention can be made in various ways, depending largely upon the formof the tag. Components of the invention and tags are preferably attachedby a covalent bond.

The production of tag-polypeptides by recombinant means when the tag isalso a polypeptide is described below. Production of FLAG (DYKDDDDK; SEQID NO:16)-labeled proteins is well known in the art and kits for suchproduction are commercially available (for example, from Kodak andSigma). Methods for the production and use of FLAG (DYKDDDDK; SEQ IDNO:16)-labeled proteins are found, for example, in Winston et al., Genesand Devel. 13:270–283 (1999), incorporated herein in its entirety, aswell as product handbooks provided with the above-mentioned kits.

Biotinylation of target molecules and substrates is well known, forexample, a large number of biotinylation agents are known, includingamine-reactive and thiol-reactive agents, for the biotinylation ofproteins, nucleic acids, carbohydrates, carboxylic acids; see chapter 4,Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated byreference. A biotinylated substrate can be attached to a biotinylatedcomponent via avidin or streptavidin. Similarly, a large number ofhaptenylation reagents are also known (Id.).

Methods for labeling of proteins with radioisotopes are known in theart. For example, such methods are found in Ohta et al., Molec. Cell3:535–541 (1999), which is hereby incorporated by reference in itsentirety.

Production of proteins having His-tags by recombinant means is wellknown, and kits for producing such proteins are commercially available.Such a kit and its use is described in the QIAexpress Handbook fromQuiagen by Joanne Crowe et al., hereby expressly incorporated byreference.

The functionalization of labels with chemically reactive groups such asthiols, amines, carboxyls, etc. is generally known in the art. In apreferred embodiment, the tag is functionalized to facilitate covalentattachment.

The covalent attachment of the tag may be either direct or via a linker.In one embodiment, the linker is a relatively short coupling moiety,that is used to attach the molecules. A coupling moiety may besynthesized directly onto a component of the invention, ubiquitin forexample, and contains at least one functional group to facilitateattachment of the tag. Alternatively, the coupling moiety may have atleast two functional groups, which are used to attach a functionalizedcomponent to a functionalized tag, for example. In an additionalembodiment, the linker is a polymer. In this embodiment, covalentattachment is accomplished either directly, or through the use ofcoupling moieties from the component or tag to the polymer. In apreferred embodiment, the covalent attachment is direct, that is, nolinker is used. In this embodiment, the component preferably contains afunctional group such as a carboxylic acid which is used for directattachment to the functionalized tag. It should be understood that thecomponent and tag may be attached in a variety of ways, including thoselisted above. What is important is that manner of attachment does notsignificantly alter the functionality of the component. For example, intag-ubiquitin, the tag should be attached in such a manner as to allowthe ubiquitin to be covalently bound to other ubiquitin to formpolyubiquitin chains. As will be appreciated by those in the art, theabove description of covalent attachment of a label and ubiquitinapplies equally to the attachment of virtually any two molecules of thepresent disclosure.

In a preferred embodiment, the tag is functionalized to facilitatecovalent attachment, as is generally outlined above. Thus, a widevariety of tags are commercially available which contain functionalgroups, including, but not limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to covalently attach the tag to asecond molecule, as is described herein. The choice of the functionalgroup of the tag will depend on the site of attachment to either alinker, as outlined above or a component of the invention. Thus, forexample, for direct linkage to a carboxylic acid group of a ubiquitin,amino modified or hydrazine modified tags will be used for coupling viacarbodiimide chemistry, for example using1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as is known in theart (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; seealso the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both ofwhich are hereby incorporated by reference). In one embodiment, thecarbodiimide is first attached to the tag, such as is commerciallyavailable for many of the tags described herein.

In a preferred embodiment, ubiquitin is in the form of tag-ubiquitin.

In a preferred embodiment, ubiquitin is in the form of tag-ubiquitin,wherein, tag is a partner of a binding pair. Preferably in thisembodiment the tag is FLAG (DYKDDDDK; SEQ ID NO:16) and the bindingpartner is anti-FLAG (DYKDDDDK; SEQ ID NO:16). Preferably in thisembodiment, a label is attached to the FLAG (DYKDDDDK; SEQ ID NO:16) byindirect labeling. Preferably, the label is a label enzyme. Mostpreferably, the label enzyme is horseradish peroxidase, which is reactedwith a fluorescent label enzyme substrate. Preferably, the label enzymesubstrate is Luminol. Alternatively, the label is a fluorescent label.

In another preferred embodiment, ubiquitin is in the form oftag-ubiquitin, wherein the tag is a fluorescent label. In a particularlypreferred embodiment, ubiquitin is in the form of tag1-ubiquitin andtag2-ubiquitin, wherein tag1 and tag2 are the members of a FRET pair. Inan alternate preferred embodiment, ubiquitin is in the form oftag1-ubiquitin and tag2-ubiquitin, wherein tag1 is a fluorescent labeland tag2 is a quencher of the fluorescent label. In either of thesepreferred embodiments, when tag1-ubiquitin and tag2-ubiquitin are boundthrough the activity of a ubiquitin ligase, preferably tag1 and tag2 arewithin 100 Å of each other, more preferable within 70 Å, still morepreferably within 50 Å, even more preferably within 40 Å, and in somecases, preferably within 30 Å or less.

In yet another preferred embodiment, ubiquitin is in the form oftag1,2-ubiquitin and tag1,3-ubiquitin, wherein tag1 is a member of abinding pair, preferably FLAG (DYKDDDDK; SEQ ID NO:16), tag2 is afluorescent label and tag3 is either a fluorescent label such that tag2and tag3 are members of a FRET pair or tag3 is a quencher of tag2.

In a preferred embodiment, one or more amino acids are added to theubiquitin sequence, using recombinant techniques as described herein, toprovide an attachment point for a tag, preferably a fluorescent label ora quencher. In a preferred embodiment, the one or more amino acids areCys or Ala-Cys. Preferably, the one or more amino acids are attached tothe N-terminal of the ubiquitin. In a preferred embodiment, the one ormore amino acids intervenes the sequence of a FLAG (DYKDDDDK; SEQ IDNO:16) tag and the ubiquitin. In a preferred embodiment, the tag,preferably a fluorescent label or a quencher, is attached to the addedCysteine.

The present invention provides methods and compositions comprisingcombining ubiquitin and E1. By “E1” is meant a ubiquitin activatingenzyme. In a preferred embodiment, E1 is capable of transferringubiquitin to an E2, defined below. In a preferred embodiment, E1 bindsubiquitin. In a preferred embodiment, E1 forms a high energy thiolesterbond with ubiquitin, thereby “activating” the ubiquitin.

In a preferred embodiment, E1 proteins useful in the invention includethose having the amino acid sequence of the polypeptide having ATCCaccession numbers A38564, S23770, AAA61246, P22314, CAA40296 andBAA33144, incorporated herein by reference. In a preferred embodiment,E1 has the amino acid sequence shown in FIG. 6B or is encoded by anucleic acid comprising the sequence shown in FIG. 6A. Preferably E1 ishuman E1. E1 is commercially available from Affiniti Research Products(Exeter, U.K.).

In a preferred embodiment, nucleic acids which may be used for producingE1 proteins for the invention include, but are not limited to, thosedisclosed by ATCC accession numbers M58028, X56976 and AB012190,incorporated herein by reference. In a preferred embodiment, E1 isencoded by a nucleic acid having a sequence consisting essentially ofthe sequence shown in FIG. 6A. Variants of the cited E1 proteins, alsoincluded in the term “E1”, can be made as described herein.

In a preferred embodiment, the compositions of the invention compriseE2. By “E2” is meant a ubiquitin carrier enzyme (also known as aubiquitin conjugating enzyme). In a preferred embodiment, ubiquitin istransferred from E1 to E2. In a preferred embodiment, the transferresults in a thiolester bond formed between E2 and ubiquitin. In apreferred embodiment, E2 is capable of transferring ubiquitin to an E3,defined below. In a preferred embodiment, the ubiquitination substrateprotein is ubiquitin.

In a preferred embodiment, proteins which may be used in the presentinvention as E2 include, but are not limited to, those having the aminoacid sequences disclosed in ATCC accession numbers AAC37534, P49427,CAA82525, AAA58466, AAC41750, P51669, AAA91460, AAA91461, CAA63538,AAC50633, P27924, AAB36017, Q16763, AAB86433, AAC26141, CAA04156,BAA11675, Q16781, NP_(—)003333, BAB18652, AAH00468, CAC16955, CAB76865,CAB76864, NP_(—)05536, O00762, XP_(—)009804, XP_(—)009488, XP_(—)006823,XP_(—)006343, XP_(—)005934, XP_(—)002869, XP_(—)003400XP_(—)009365,XP_(—)010361, XP_(—)004699, XP_(—)004019, O14933, P27924, P50550,P52485, P51668, P51669, P49459, P37286, P23567, P56554, and CAB45853,each of which is incorporated herein by reference. Particularlypreferred are sequences disclosed in ATCC accession numbers NP003331,NP003330, NP003329, P49427, AAB53362, NP008950, XP009488and AAC41750,also incorporated by reference. The skilled artisan will appreciate thatmany different E2 proteins and isozymes are known in the filed and maybe used in the present invention, provided that the E2 has ubiquitinconjugating activity. Also specifically included within the term “E2”are variants of E2, which can be made as described herein.

In a preferred embodiment, E2 is one of Ubc5 (Ubch5, preferably Ubch5c),Ubc3 (Ubch3), Ubc4 (Ubch4) and UbcX (Ubc10, Ubch10). In a preferredembodiment, E2 is Ubc5c. In a preferred embodiment, E2 has the aminoacid sequence shown in FIG. 7B or is encoded by a nucleic acidconsisting essentially of the sequence shown in FIG. 7A.

In a preferred embodiment, nucleic acids which may be used to make E2include, but are not limited to, those nucleic acids having sequencesdisclosed in ATCC accession numbers L2205, Z29328, M92670, L40146,U39317, U39318, X92962, U58522, S81003, AF031141, AF075599, AJ000519,XM009488, NM007019, U73379, L40146 and D83004, each of which isincorporated herein by reference. As described above, variants of theseand other E2 encoding nucleic acids may also be used to make variant E2proteins.

In a preferred embodiment, the nucleic acid used to make E2 comprisesthe sequence shown in FIG. 7A.

In a preferred embodiment, E2 has a tag, as defined above, with thecomplex being referred to herein as “tag-E2”. Preferred E2 tags include,but are not limited to, labels, partners of binding pairs and substratebinding elements. In a most preferred embodiment, the tag is a His-tagor GST-tag.

The present invention provides methods and compositions comprising E3.By “E3” is meant a ubiquitin ligase, as defined above, comprising one ormore components associated with ligation of ubiquitin to aubiquitination substrate protein for ubiquitin-dependent proteolysis. Ina preferred embodiment, E3 comprises a ring finger protein and a Cullin.In a preferred embodiment, RING finger proteins include, but are notlimited to, ROC1, ROC2 and APC11. In a preferred embodiment, Cullinsinclude, but are not limited to, CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5and APC2.

In a preferred embodiment, RING finger proteins include, but are notlimited to, proteins having the amino acid sequence disclosed in ATCCaccession numbers AAD30147 and AAD30146 and 6320196, incorporated hereinby reference. In a more preferred embodiment, the ring finger proteinhas a sequence selected from the group consisting of that shown in FIG.8, FIG. 9 and FIG. 10B.

In a preferred embodiment, Cullins include, but are not limited to,proteins having the amino acid sequences disclosed in ATCC accessionnumbers 4503161, AAC50544, AAC36681, 4503163, AAC51190, AAD23581,4503165, AAC36304, AAC36682, AAD45191, AAC50548, Q13620, 4503167 andAAF05751, each of which is incorporated herein by reference. Inaddition, in the context of the invention, each of the RING fingerproteins and Cullins encompass variants of the known or listedsequences, as described herein.

In a preferred embodiment, the Cullin has a sequence as shown in FIG.11B or 12B.

These E3 proteins and variants may be made as described herein. In apreferred embodiment, nucleic acids used to make the RING fingerproteins include, but are not limited to, those having the nucleic acidsequences disclosed in ATCC accession numbers AF142059, AF142060 andnucleic acids 433493 to 433990 of NC 001136. In a preferred embodiment,Cullins are made from nucleic acids including, but not limited to, thosehaving nucleic acid sequences disclosed in ATCC accession numbers NM003592, U58087, AF062536, AF126404, NM 003591, U83410, NM 003590,AB014517, AF062537, AF064087, AF077188, U58091, NM 003478, X81882 andAF191337, each of which is incorporated herein by reference. Asdescribed above, variants of these sequences are also encompassed by theinvention.

In a preferred embodiment, nucleic acid used to produce ROC2 comprisesthe sequence depicted in FIG. 12A. In a preferred embodiment, nucleicacid used to produce CUL5 comprises the sequence depicted in FIG. 13A.In a preferred embodiment, nucleic acid used to produce APC2 comprisesthe sequence depicted in FIG. 14A.

In a preferred embodiment, E3 comprises the RING finger protein/Cullincombination APC11/APC2. In another preferred embodiment, E3 comprisesthe RING finger protein/Cullin combination ROC1/CUL1. In yet preferredembodiment, E3 comprises the RING finger protein/Cullin combinationROC1/CUL2. In still another preferred embodiment, E3 comprises the RINGfinger protein/Cullin combination ROC2/CUL5. However, the skilledartisan will appreciate that any combination of E3 components may beproduced and used in the invention described herein.

In an alternate embodiment, E3 comprises the ligase E3-alpha, E3A(E6-AP), HERC2, SMURF1, TRAF6, MDM2, Cbl, Sina/Siah, Itchy, IAP orNEDD-4. In this embodiment, the ligase has the amino acid sequence ofthat disclosed in ATCC accession number AAC39845, Q05086, CAA66655,CAA66654, CAA66656, AAD08657, NP_(—)002383, XP_(—)006284, AAC51970,XP_(—)013050, BAB39389, Q00987, AAF08298 or P46934, each of which isincorporated herein by reference. As above, variants are alsoencompassed by the invention. Nucleic acids for making E3 for thisembodiment include, but are not limited to, those having the sequencesdisclosed in ATCC accession numbers AF061556, XM006284, U76247,XM013050, X898032, X98031, X98033, AF071172, Z12020, AB056663, AF199364and D42055 and variants thereof.

E3 may also comprise other components, such as SKP1 and F-box proteins.The amino acid and nucleic acid sequences for SKP1 are found in ATCCaccession numbers AAC50241 and U33760, respectively. Many F-box proteinsare known in the art and their amino acid and nucleic acid sequences arereadily obtained by the skilled artisan from various published sources.

In a preferred embodiment, the E3 components are produced recombinantly,as described herein.

In a preferred embodiment, the E3 components are co-expressed in thesame host cell. Co-expression may be achieved by transforming the cellwith a vector comprising nucleic acids encoding two or more of the E3components, or by transforming the host cell with separate vectors, eachcomprising a single component of the desired E3 protein complex. In apreferred embodiment, the RING finger protein and Cullin are expressedin a single host transfected with two vectors, each comprising nucleicacid encoding one or the other polypeptide, as described in furtherdetail in the Examples.

In a preferred embodiment, E3 has a tag, which complex is referred toherein as “tag-E3”. Preferably, the tag is attached to only onecomponent of the E3. Preferred E3 tags include, but are not limited to,labels, partners of binding pairs and substrate binding elements. Morepreferably, the tag is a surface substrate binding molecule. Mostpreferably, the tag is a His-tag or GST-tag.

In an embodiment herein, ubiquitin and ubiquitination enzymes and theircomponents are cloned and expressed as outlined below. Thus, probe ordegenerate polymerase chain reaction (PCR) primer sequences may be usedto find other related ubiquitination proteins from humans or otherorganisms. As will be appreciated by those in the art, particularlyuseful probe and/or PCR primer sequences include the unique areas of anucleic acid sequence. As is generally known in the art, preferred PCRprimers are from about 15 to about 35 nucleotides in length, with fromabout 20 to about 30 being preferred, and may contain inosine as needed.The conditions for the PCR reaction are well known in the art. It istherefore also understood that provided along with the sequences in thesequences cited herein are portions of those sequences, wherein uniqueportions of 15 nucleotides or more are particularly preferred. Theskilled artisan can routinely synthesize or cut a nucleotide sequence tothe desired length.

Once isolated from its natural source, e.g., contained within a plasmidor other vector or excised therefrom as a linear nucleic acid segment,the recombinant nucleic acid can be further-used as a probe to identifyand isolate other nucleic acids. It can also be used as a “precursor”nucleic acid to make modified or variant nucleic acids and proteins.

Using the nucleic acids of the present invention which encode a protein,a variety of expression vectors are made. The expression vectors may beeither self-replicating extrachromosomal vectors or vectors whichintegrate into a host genome. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the protein. The term“control sequences” refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. As another example, operablylinked refers to DNA sequences linked so as to be contiguous, and, inthe case of a secretory leader, contiguous and in reading fram. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the protein; for example, transcriptional andtranslational regulatory nucleic acid sequences from Bacillus arepreferably used to express the protein in Bacillus. Numerous types ofappropriate expression vectors, and suitable regulatory sequences areknown in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

A preferred expression vector system is a retroviral vector system suchas is generally described in PCT/US97/01019 and PCT/US97/01048, both ofwhich are hereby expressly incorporated by reference.

Proteins of the present invention are produced by culturing a host celltransformed with an expression vector containing nucleic acid encodingthe protein, under the appropriate conditions to induce or causeexpression of the protein. The conditions appropriate for proteinexpression will vary with the choice of the expression vector and thehost cell, and will be easily ascertained by one skilled in the artthrough routine experimentation. For example, the use of constitutivepromoters in the expression vector will require optimizing the growthand proliferation of the host cell, while the use of an induciblepromoter requires the appropriate growth conditions for induction. Inaddition, in some embodiments, the timing of the harvest is important.For example, the baculoviral systems used in insect cell expression arelytic viruses, and thus harvest time selection can be crucial forproduct yield.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Drosophila melanogaster cells, Pichia pastoris and P.methanolica, Saccharomyces cerevisiae and other yeasts, E. coli,Bacillus subtilis, SF9 cells, SF21 cells, C129 cells, Saos-2 cells, Hi-5cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells. Of greatestinterest are Pichia pastoris and P. methanolica, E. coli, SF9 cells,SF21 cells and Hi-5 cells.

In a preferred embodiment, the proteins are expressed in mammaliancells. Mammalian expression systems are also known in the art, andinclude retroviral systems. A mammalian promoter is any DNA sequencecapable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for a protein intoMRNA. A promoter will have a transcription initiating region, which isusually placed proximal to the 5′ end of the coding sequence, and a TATAbox, using a located 25–30 base pairs upstream of the transcriptioninitiation site. The TATA box is thought to direct RNA polymerase II tobegin RNA synthesis at the correct site. A mammalian promoter will alsocontain an upstream promoter element (enhancer element), typicallylocated within 100 to 200 base pairs upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation. Of particular use asmammalian promoters are the promoters from mammalian viral genes, sincethe viral genes are often highly expressed and have a broad host range.Examples include the SV40 early promoter, mouse mammary tumor virus LTRpromoter, adenovirus major late promoter, herpes simplex virus promoter,and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature MRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenylation signals includethose derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In a preferred embodiment, proteins are expressed in bacterial systems.Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3′)transcription of the coding sequence of a protein into mRNA. A bacterialpromoter has a transcription initiation region which is usually placedproximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. Sequences encoding metabolic pathwayenzymes provide particularly useful promoter sequences. Examples includepromoter sequences derived from sugar metabolizing enzymes, such asgalactose, lactose and maltose, and sequences derived from biosyntheticenzymes such as tryptophan.

Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. coli, the ribosome binding site iscalled the Shine-Delgarno (SD) sequence and includes an initiation codonand a sequence 3–9 nucleotides in length located 3–11 nucleotidesupstream of the initiation codon.

The expression vector may also include a signal peptide sequence thatprovides for secretion of the protein in bacteria. The signal sequencetypically encodes a signal peptide comprised of hydrophobic amino acidswhich direct the secretion of the protein from the cell, as is wellknown in the art. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic space, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterial hostcells using techniques well known in the art, such as calcium chloridetreatment, electroporation, and others.

In one embodiment, proteins are produced in insect cells. Expressionvectors for the transformation of insect cells, and in particular,baculovirus-based expression vectors, are well known in the art.

In a preferred embodiment, proteins are produced in yeast cells. Yeastexpression systems are well known in the art, and include expressionvectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa,Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichiaguillerimondii P. methanolica and P. pastoris, Schizosaccharomycespombe, and Yarrowia lipolytica. Preferred promoter sequences forexpression in yeast include the inducible GAL1,10 promoter, thepromoters from alcohol dehydrogenase, enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase,hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvatekinase, and the acid phosphatase gene. Yeast selectable markers includeADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance totunicamycin; the neomycin phosphotransferase gene, which confersresistance to G418; and the CUP1 gene, which allows yeast to grow in thepresence of copper ions.

The protein may also be made as a fusion protein, using techniques wellknown in the art. Thus, for example, the protein may be made as a fusionprotein to increase expression, or for other reasons. For example, whenthe protein is a peptide, the nucleic acid encoding the peptide may belinked to other nucleic acid for expression purposes. Similarly,proteins of the invention can be linked to protein labels, such as greenfluorescent protein (GFP), red fluorescent protein (RFP), bluefluorescent protein (BFP), yellow fluorescent protein (YFP), etc.

In a preferred embodiment, the protein is purified or isolated afterexpression. Proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the ubiquitin protein may be purified using a standard anti-ubiquitinantibody column. Ultrafiltration and diafiltration techniques, inconjunction with protein concentration, are also useful. For generalguidance in suitable purification techniques, see Scopes, R., ProteinPurification, Springer-Verlag, NY (1982). The degree of purificationnecessary will vary depending on the use of the protein. In someinstances no purification will be necessary.

Once made, the compositions find use in a number of applications,including, but not limited to, screens for modulators of ubiquitination.By “modulator” is meant a compound which can increase or decreaseubiquitination. The skilled artisan will appreciate that modulators ofubiquitination may affect enzyme activity, enzyme interaction with thesubstrate, interaction between ubiquitin and the substrate, or acombination of these. A modulator that specifically affects ubiquitinligase activity is a ubiquitin ligase modulator.

By “candidate”, “candidate agent”, “candidate modulator”, “candidateubiquitination modulator” or grammatical equivalents herein is meant anymolecule, e.g. proteins (which herein includes proteins, polypeptides,and peptides), small organic or inorganic molecules, polysaccharides,polynucleotides, etc. which are to be tested for ubiquitinationmodulator activity. Candidate agents encompass numerous chemicalclasses. In a preferred embodiment, the candidate agents are organicmolecules, particularly small organic molecules, comprising functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more chemical functional groups.

Candidate modulators are obtained from a wide variety of sources, aswill be appreciated by those in the art, including libraries ofsynthetic or natural compounds. As will be appreciated by those in theart, the present invention provides a rapid and easy method forscreening any library of candidate modulators, including the widevariety of known combinatorial chemistry-type libraries.

In a preferred embodiment, candidate modulators are synthetic compounds.Any number of techniques are available for the random and directedsynthesis of a wide variety of organic compounds and biomolecules,including expression of randomized oligonucleotides. See for example WO94/24314, hereby expressly incorporated by reference, which discussesmethods for generating new compounds, including random chemistry methodsas well as enzymatic methods. As described in WO 94/24314, one of theadvantages of the present method is that it is not necessary tocharacterize the candidate modulator prior to the assay; only candidatemodulators that increase or decease ubiquitin ligase activity need beidentified. In addition, as is known in the art, coding tags using splitsynthesis reactions may be done, to essentially identify the chemicalmoieties tested.

Alternatively, a preferred embodiment utilizes libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsthat are available or readily produced.

Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means. Known pharmacological agents may be subjected todirected or random chemical modifications, including enzymaticmodifications, to produce structural analogs.

In a preferred embodiment, candidate modulators include proteins,nucleic acids, and chemical moieties.

In a preferred embodiment, the candidate modulator are proteins, asdefined above. In a preferred embodiment, the candidate modulators arenaturally occurring proteins or fragments of naturally occurringproteins. Thus, for example, cellular extracts containing proteins, orrandom or directed digests of proteinaceous cellular extracts, may betested, as is more fully described below. In this way libraries ofprocaryotic and eucaryotic proteins may be made for screening againstany number of ubiquitin ligase compositions. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the candidate modulators are peptides of fromabout 2 to about 50 amino acids, with from about 5 to about 30 aminoacids being preferred, and from about 8 to about 20 being particularlypreferred. The peptides may be digests of naturally occurring proteinsas is outlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

The library should provide a sufficiently structurally diversepopulation of randomized agents to effect a probabilistically sufficientrange of diversity to allow interaction with a particular ubiquitinligase enzyme. Accordingly, an interaction library must be large enoughso that at least one of its members will have a structure that interactswith a ubiquitin ligase enzyme. Although it is difficult to gauge therequired absolute size of an interaction library, nature provides a hintwith the immune response: a diversity of 10⁷–10⁸ different antibodiesprovides at least one combination with sufficient affinity to interactwith most potential antigens faced by an organism. Published in vitroselection techniques have also shown that a library size of 10⁷ to 10⁸is sufficient to find structures with affinity for a target. A libraryof all combinations of a peptide 7 to 20 amino acids in length, such asgenerally proposed herein, has the potential to code for 20⁷ (10⁹) to20²⁰. Thus, with libraries of 10⁷ to 10⁸ different molecules the presentmethods allow a “working” subset of a theoretically complete interactionlibrary for 7 amino acids, and a subset of shapes for the 20²⁰ library.Thus, in a preferred embodiment, at least 10⁶, preferably at least 10⁷,more preferably at least 10⁸ and most preferably at least 10⁹ differentsequences are simultaneously analyzed in the subject methods. Preferredmethods maximize library size and diversity.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bias is towards peptides or nucleic acidsthat interact with known classes of molecules. For example, when thecandidate modulator is a peptide, it is known that much of intracellularsignaling is carried out via short regions of polypeptides interactingwith other polypeptides through small peptide domains. For instance, ashort region from the HIV-1 envelope cytoplasmic domain has beenpreviously shown to block the action of cellular calmodulin. Regions ofthe Fas cytoplasmic domain, which shows homology to the mastoparan toxinfrom Wasps, can be limited to a short peptide region with death-inducingapoptotic or G protein inducing functions. Magainin, a natural peptidederived from Xenopus, can have potent anti-tumor and anti-microbialactivity. Short peptide fragments of a protein kinase C isozyme (βPKC),have been shown to block nuclear translocation of βPKC in Xenopusoocytes following stimulation. And, short SH-3 target peptides have beenused as psuedosubstrates for specific binding to SH-3 proteins. This isof course a short list of available peptides with biological activity,as the literature is dense in this area. Thus, there is much precedentfor the potential of small peptides to have activity on intracellularsignaling cascades. In addition, agonists and antagonists of any numberof molecules may be used as the basis of biased randomization ofcandidate modulators as well.

Thus, a number of molecules or protein domains are suitable as startingpoints for the generation of biased randomized candidate modulators. Alarge number of small molecule domains are known, that confer a commonfunction, structure or affinity. In addition, as is appreciated in theart, areas of weak amino acid homology may have strong structuralhomology. A number of these molecules, domains, and/or correspondingconsensus sequences, are known, including, but are not limited to, SH-2domains, SH-3 domains, Pleckstrin, death domains, proteasecleavage/recognition sites, enzyme inhibitors, enzyme substrates, Traf,etc.

In a preferred embodiment, the candidate modulators are nucleic acids.With reference to candidate modulators, by “nucleic acid” or“oligonucleotide” or grammatical equivalents herein means at least twonucleotides covalently linked together. A nucleic acid of the presentinvention will generally contain phosphodiester bonds, although in somecases, as outlined below, nucleic acid analogs are included that mayhave alternate backbones, comprising, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J.Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169–176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labels, or to increase thestability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. As used herein, the term“nucleoside” includes nucleotides and nucleoside and nucleotide analogs,and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus for example the individual units of a peptide nucleicacid, each containing a base, are referred to herein as a nucleoside.

As described above generally for proteins, nucleic acid candidatemodulator may be naturally occurring nucleic acids, random nucleicacids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eucaryotic genomes may be used as is outlined above forproteins. Where the ultimate expression product is a nucleic acid, atleast 10, preferably at least 12, more preferably at least 15, mostpreferably at least 21 nucleotide positions need to be randomized, withmore preferable if the randomization is less than perfect. Similarly, atleast 5, preferably at least 6, more preferably at least 7 amino acidpositions need to be randomized; again, more are preferable if therandomization is less than perfect.

In a preferred embodiment, the candidate modulators are organicmoieties. In this embodiment, as is generally described in WO 94/24314,candidate agents are synthesized from a series of substrates that can bechemically modified. “Chemically modified” herein includes traditionalchemical reactions as well as enzymatic reactions. These substratesgenerally include, but are not limited to, alkyl groups (includingalkanes, alkenes, alkynes and heteroalkyl), aryl groups (includingarenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones,acids, esters, amides, cyclic compounds, heterocyclic compounds(including purines, pyrimidines, benzodiazepins, beta-lactams,tetracylines, cephalosporins, and carbohydrates), steroids (includingestrogens, androgens, cortisone, ecodysone, etc.), alkaloids (includingergots, vinca, curare, pyrollizdine, and mitomycines), organometalliccompounds, hetero-atom bearing compounds, amino acids, and nucleosides.Chemical (including enzymatic) reactions may be done on the moieties toform new substrates or candidate agents which can then be tested usingthe present invention.

As will be appreciated by those in the art, it is possible to screenmore than one type of candidate modulator at a time. Thus, the libraryof candidate modulators used may include only one type of agent (i.e.peptides), or multiple types (peptides and organic agents). The assay ofseveral candidates at one time is further discussed below.

The present invention provides methods and compositions comprisingcombining several components. In a preferred embodiment, a preferredcombination is tag-ubiquitin, E1, E2, and E3. Preferably the tag is alabel, a partner of a binding pair, or a substrate binding molecule.More preferably, the tag is a fluorescent label or a binding pairpartner. In a preferred embodiment, the tag is a binding pair partnerand the ubiquitin is labeled by indirect labeling. In the indirectlabeling embodiment, preferably the label is a fluorescent label or alabel enzyme. In an embodiment comprising a label enzyme, preferably thesubstrate for that enzyme produces a fluorescent product. In a preferredembodiment, the label enzyme substrate is luminol. In a preferredembodiment, combining specifically excludes combining the componentswith a target protein.

In another preferred embodiment, a preferred combination isTag1-ubiquitin, tag2-ubiquitin, E1, E2 and E3. Preferably, tag1 and tag2are labels, preferably fluorescent labels, most preferably tag1 and tag2constitute a FRET pair.

In a preferred embodiment, a preferred combination is tag1-ubiquitin,E1, E2 and tag2-E3. Preferably, tag1 is a label, a partner of a bindingpair, or a substrate binding molecule and tag2 is a different label,partner of a binding pair, or substrate binding molecule. Morepreferably, tag1 is a fluorescent label or a member of a binding pair.When tag1 is a member of a binding pair, preferably tag1 is indirectlylabeled. Still more preferably, tag-1 is indirectly labeled with a labelenzyme. Preferably the label enzyme substrate used to reveal thepresence of the enzyme produces a fluorescent product, and morepreferably is luminol. In the presently described combination,preferably tag2 is a surface substrate binding element, more preferablya His-tag.

In a preferred embodiment, a preferred combination is tag1-ubiquitin, E1and tag2-E2. In this embodiment, preferably, tag1 is a label, a partnerof a binding pair, or a substrate binding molecule and tag2 is adifferent label, partner of a binding pair, or substrate bindingmolecule. More preferably, tag-1 is a label or a member of a bindingpair. When tag1 is a member of a binding pair, preferably tag1 isindirectly labeled. In a preferred embodiment, the tag1 label (direct orindirect) is a fluorescent label or a label enzyme. When the tag1 label(direct or indirect) is a label enzyme, preferably the reactionsubstrate used to reveal the presence of the enzyme produces afluorescent product, and more preferably is luminol. In the presentlydescribed combination, preferably tag2 is a substrate binding element,more preferably a His-tag.

In a preferred embodiment, the compositions of the invention do notcomprise a target protein. In this embodiment, ubiquitin is the soleubiquitination substrate, as discussed above. This embodiment stands incontrast to previous ubiquitination enzyme assays, which requiredaddition of a target protein as part of the composition. Because thedifferent combinations of E3 and E2 and combinations of E3 subunits arespecific to particular target proteins, the present assays are much moreversatile, allowing any variation of such combinations without firstidentifying the specific target protein to which the combination isdirected.

The components of the present compositions may be combined in varyingamounts. In a preferred embodiment, ubiquitin is combined at a finalconcentration of from 20 to 200 ng per 100 μl reaction solution, mostpreferable at about 100 ng per 100 μl reaction solution.

In a preferred embodiment, E1 is combined at a final concentration offrom 1 to 50 ng per 100 μl reaction solution, more preferably from 1 ngto 20 ng per 100 μl reaction solution, most preferably from 5 ng to 10ng per 100 μl reaction solution.

In a preferred embodiment, E2 is combined at a final concentration of 10to 100 ng per 100 μl reaction solution, more preferably 10–50 ng per 100μl reaction solution.

In a preferred embodiment, E3 is combined at a final concentration offrom 1 ng to 500 ng per 100 μl reaction solution, more preferably from50 to 400 ng per 100 μl reaction solution, still more preferably from100 to 300 ng per 100 μl reaction solution, most preferably about 100 ngper 100 μl reaction solution.

The components of the invention are combined under reaction conditionsthat favor ubiquitin ligase activity and/or ubiquitination activity.Generally, this will be physiological conditions. Incubations may beperformed at any temperature which facilitates optimal activity,typically between 4 and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapid highthrough put screening. Typically between 0.5 and 1.5 hours will besufficient.

A variety of other reagents may be included in the compositions. Theseinclude reagents like salts, solvents, buffers, neutral proteins, e.g.albumin, detergents, etc. which may be used to facilitate optimalubiquitination enzyme activity and/or reduce non-specific or backgroundinteractions. Also reagents that otherwise improve the efficiency of theassay, such as protease inhibitors, nuclease inhibitors, anti-microbialagents, etc., may be used. The compositions will also preferably includeadenosine tri-phosphate (ATP).

The mixture of components may be added in any order that promotesubiquitin ligase activity or optimizes identification of candidatemodulator effects. In a preferred embodiment, ubiquitin is provided in areaction buffer solution, followed by addition of the ubiquitinationenzymes. In an alternate preferred embodiment, ubiquitin is provided ina reaction buffer solution, a candidate modulator is then added,followed by addition of the ubiquitination enzymes.

Once combined, preferred methods of the invention comprise measuring theamount of ubiquitin bound to E3. In an alternate preferred embodiment inwhich the combination lacks E3, preferred methods of the inventioncomprise measuring the amount of ubiquitin bound to E2. As will beunderstood by one of ordinary skill in the art, the mode of measuringwill depend on the specific tag attached to the ubiquitin. As will alsobe apparent to the skilled artisan, the amount of ubiquitin bound willencompass not only the particular ubiquitin protein bound directly tothe ubiquitination enzyme, but also the ubiquitin proteins bound to thatparticular ubiquitin in a polyubiquitin chain.

In a preferred embodiment, the tag attached to the ubiquitin is afluorescent label. In a preferred embodiment, the tag attached toubiquitin is an enzyme label or a binding pair member which isindirectly labeled with an enzyme label. In this latter preferredembodiment, the enzyme label substrate produces a fluorescent reactionproduct. In these preferred embodiments, the amount of ubiquitin boundis measured by luminescence.

As used herein, “luminescence” or “fluorescent emission” means photonemission from a fluorescent label. In an embodiment where FRET pairs areused, fluorescence measurements may be taken continuously or attime-points during the ligation reaction. Equipment for such measurementis commercially available and easily used by one of ordinary skill inthe art to make such a measurement.

Other modes of measuring bound ubiquitin are well known in the art andeasily identified by the skilled artisan for each of the labelsdescribed herein. For instance, radioisotope labeling may be measured byscintillation counting, or by densitometry after exposure to aphotographic emulsion, or by using a device such as a PhosphorImager.Likewise, densitometry may be used to measure bound ubiquitin followinga reaction with an enzyme label substrate that produces an opaqueproduct when an enzyme label is used.

In preferred methods of the present invention, E3 is bound to a surfacesubstrate. This may be done directly, as described above for the bindingof a label to ubiquitin. This may also be accomplished using tag-E3,wherein the tag is a surface substrate binding molecule.

In another preferred embodiment of the invention, E2 is bound to asurface substrate in the absence of E3. This may be done directly, asdescribed above for the binding of a label to ubiquitin. This may alsobe accomplished using tag-E2, wherein the tag is a surface substratebinding molecule.

In the two preferred embodiments described immediately above, E3 and E2are in the form of tag-E3 and tag-E2, respectively, and are bound to asurface substrate via a surface substrate binding molecule tag. Ingeneral, any substrate binding molecule can be used. In a preferredembodiment, the tag is a His-tag and the surface substrate is nickel. Ina preferred embodiment, the nickel surface substrate is present on thesurface of the wells of a multi-well plate, such as a 96 well plate.Such multi-well plates are commercially available. The binding of theenzyme to a surface substrate facilitates the separation of boundubiquitin from unbound ubiquitin. In the present embodiment, the unboundubiquitin is easily washed from the receptacle following the ligationreaction. As will be appreciated by those of skill in the art, the useof any surface substrate binding element and receptacle having thesurface substrate to which it binds will be effective for facilitatingthe separation of bound and unbound ubiquitin.

In an alternative embodiment, E3 or E2 is bound, directly or via asubstrate binding element, to a bead. Following ligation, the beads maybe separated from the unbound ubiquitin and the bound ubiquitinmeasured. In a preferred embodiment, E3 or E2 is bound to beads and thecomposition used includes tag-ubiquitin wherein tag is a fluorescentlabel. In this embodiment, the beads with bound ubiquitin may beseparated using a fluorescence-activated cell sorting (FACS) machine.Methods for such use are described in U.S. patent application Ser. No.09/047,119, which is hereby incorporated in its entirety. The amount ofbound ubiquitin can then be measured.

In another embodiment, none of the ubiquitination enzymes is bound to asubstrate. Preferably in this embodiment, the composition comprisestag1-ubiquitin, tag2-ubiquitin, E1, E2 and E3. Preferably, tag1 and tag2are labels, preferably fluorescent labels, most preferably tag1 and tag2constitute a FRET pair. In this embodiment, ubiquitination is measuredby measuring the fluorescent emission spectrum. This measuring may becontinuous or at one or more times following the combination of thecomponents. Alteration in the fluorescent emission spectrum of thecombination as compared with unligated ubiquitin indicates the amount ofubiquitination. The skilled artisan will appreciate that in thisembodiment, alteration in the fluorescent emission spectrum results fromubiquitin bearing different members of the FRET pair being brought intoclose proximity, either through the formation of poly-ubiquitin and/orby binding nearby locations on a protein, preferably a target protein.

In a preferred embodiment, the compositions of the invention are used toidentify ubiquitination modulators. In this embodiment, the compositionincludes a candidate modulator. In a preferred embodiment, the measuredamount and/or rate of tag-ubiquitin binding to E3 is compared with thethat when the candidate modulator is absent from the composition,whereby the presence or absence of the modulators effect on ubiquitinligase activity is determined. In this embodiment, whether the modulatorenhances or inhibits ubiquitination is also determined.

In a preferred embodiment, the composition of the invention containing acandidate modulator lacks E3 and the amount and/or rate of ubiquitinbinding to E2 is measured. This embodiment may also comprise the step ofcomparing the amount and/or rate of ubiquitin binding to E2 in acomposition lacking both E3 and the candidate modulator, whereby themodulatory activity of the candidate on ubiquitination enzymes otherthan E3 is determined. In a preferred embodiment, the percentagedifference in the amount of ubiquitin bound to E2 in the presence andabsence of the candidate modulator is compared with the percentagedifference in the amount bound to E3 in the presence and absence ofcandidate modulator, whereby the point of effect of the candidatemodulator in the enzyme cascade is determined. That is, it is determinedwhether the candidate modulator affects E3 ubiquitin ligase activity orit affects E1 ubiquitin activating activity and/or E2 ubiquitinconjugating activity.

In another preferred embodiment, the compositions of the invention areused to identify ubiquitination modulators. In this embodiment, thecomposition includes a candidate modulator. In a preferred embodiment,where tag1 and tag2 constitute a FRET pair, the measured amount and/orrate of tag1-ubiquitin and tag2-ubiquitin binding to a substrate protein(as poly-ubiquitin and/or ubiquitin bound to a target protein) iscompared with the amount or rate of such binding in the absence of thecandidate modulator, whereby the presence or absence of the modulator'seffect on ubiquitin ligase activity is determined. In this embodiment,whether the modulator enhances or inhibits ubiquitination is alsodetermined.

In a preferred embodiment, multiple assays are performed simultaneouslyin a high throughput screening system. In this embodiment, multipleassays may be performed in multiple receptacles, such as the wells of a96 well plate or other multi-well plate. As will be appreciated by oneof skill in the art, such a system may be applied to the assay ofmultiple candidate modulators and/or multiple combination of E3components and/or E2–E3 pairings. In a preferred embodiment, the presentinvention is used in a high-throughput screening system for determiningthe ubiquitin ligase activity of different E2–E3 pairings and/ordifferent E3 component combinations. In an alternate preferredembodiment, the present invention is used in a high throughput screeningsystem for simultaneously testing the effect of individual candidatemodulators.

In another aspect, the invention provides a method of assaying forubiquitination activity or ubiquitination enzyme activity in a mixture.Ubiquitin is introduced into a cell or mixture of protein, preferably acell lysate, under conditions in which ubiquitination can take place. Inthis embodiment, the ubiquitin is in the form of tag1-ubiquitin andtag2-ubiquitin, wherein tag1 and tag2 constitute a FRET pair or tag1 isa fluorescent label and tag2 is a quencher of tag1. Fluorescent emissionspectrum is measured as an indication of whether ubiquitination activityis present in the mixture or cell. In a preferred embodiment, theubiquitin also comprises a member of a binding pair, such as FLAG(DYKDDDDK; SEQ ID NO:16). In this latter embodiment, components involvedin ubiquitination can be isolated from the mixture using any one of anumber of affinity-based separation means such as fluorescent beadscoated with anti-FLAG (DYKDDDDK; SEQ ID NO:16) antibody or aminoprecipitation using anti-FLAG (DYKDDDDK; SEQ ID NO:16) antibodies, orusing anti-FLAG (DYKDDDDK; SEQ ID NO:16) antibody attached to a solidsupport. Other means of separating ubiquitin bound components of thecell or mixture will be readily apparent to the skilled artisan.Ubiquitin bound components so separated in this method may include E3and target protein. The skilled artisan will appreciate that separationof-these components for individual identification or subsequentinvestigation may be obtained by several means well known in the art,such as by HPLC or electrophoresis.

It is understood by the skilled artisan that the steps of the assaysprovided herein can vary in order. It is also understood, however, thatwhile various options (of compounds, properties selected or order ofsteps) are provided herein, the options are also each providedindividually, and can each be individually segregated from the otheroptions provided herein. Moreover, steps which are obvious and known inthe art that will increase the sensitivity of the assay are intended tobe within the scope of this invention. For example, there may beadditionally washing steps, blocking steps, etc.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are expressly incorporated by reference intheir entirety.

EXAMPLES Example 1 Production of E2, E3 and Ubiquitin

E2 Production

The open reading frame of E2 (Ubc5C) was amplified by PCR and clonedinto the pGex-6p-1 E. Coli. expression vector (Amersham Pharmacia) asBg1II-EcoRI fragments, with N-terminus in frame fused to the GST-tag.

Materials and Methods

Plasmid is transformed in BL21 DE3 competent E.coli (Stratagene, cat #230132). Cells are grown at 37° C. in TB+100 ug/ml ampicillin and 0.4%glucose to an OD600 of about 0.6, induced with addition of 320 uM IPTGand allowed to grow for another 3 h before harvest. The pellets arewashed once with cold PBS, then resuspended in about 6 volumes of lysisbuffer (20 mM Tris, 10% glycerol, 0.5 M Nacl, 2.5 mM EDTA, 1 mM TCEPplus Complete -EDTA Free Protease inhibitor tablets, 1 tablet/25 ml ofresuspended cells, pH 8.0). The suspension is homogenized and sonicated3×30 sec. NP40, then added to a final concentration of 0.5% and thetubes are rocked for 30 min at 4° C. Following centrifugation at 11000rpm for 25 to 30 min, the supernatant is incubated with GlutathioneSepharose 4B (Amersham, cat # 17-0756-01) at a ratio of 1 ml of beadsper 100 ml of original culture volume for 1 to 2 hours at 4° C. withgentle rocking. The beads are pelleted and washed once with 10 bedvolumes of the lysis buffer, then twice with 10 bed volumes ofPrescission Protease buffer (50 mM Tris-HCL, 150 mM NaCl, 1 mM EDTA, 1mM DTT, 0.1% NP-40, pH 7.0.). Prescission Protease (Amersham, product #27-0843) is added at a ratio of 80 ul (160 Units) per ml of GST resin,and allowed to incubate for 4 h at 4° C. The supernatant containing thecleaved E2 protein is collected, and the resin is washed twice with onebed volume of Prescission buffer. All three fractions are analyzed bySDS-PAGE and pooled when appropriate.

Ubiquitin Production

Ubiquitin was cloned into the pFlag-Mac Expression Vector (Sigma) as aHindIII-EcoRI fragment by PCR. This results in expression ofamino-terminal FLAG (DYKDDDDK; SEQ ID NO:16) fusion ubiquitin in E.coli.

Materials and Methods

The induction of protein expression and cell lysis is similar to theabove GST-E2 preparation, except that the supematant is loaded over aFLAG (DYKDDDDK; SEQ ID NO:16)-affinity resin (VWR, cat #1B13020) at aratio of 15m1 of beads per 1 L of original culture. The resin is thenwashed with 10 bed volumes of lysis buffer. The protein is eluted fromthe column with: 100 mM Acetic acid, 10% glycerol, 200 mM NaCl, 2.5 mMEDTA, 0.1% NP-40, pH 3.5. The elutions are collected as 1 bed volumefractions into tubes that contain 1/10^(th) volume of 2 M Tris, 80 mMB-ME, pH 9.0 to neutralize the pH. The elution fractions are analyzed bySDS-PAGE and the appropriate fractions are pooled and dialyzed against400 volumes of 20 mM Tris, 10% glycerol, 200 mM NaCl, 2.5 mM EDTA, pH8.0.

Production of E3

Coding sequences for E3 complex were also amplified by PCR andbaculoviruses were generated using the Bac-to-Bac system (GibcoBRL). E3contains two subunits, which are expressed by co-infection of the twobaculovirus in the same Hi-5 insect cells. One of the subunit isHis-tagged, with the other associating subunit untagged.

The detail procedure was done following the Bac to Bac BaculovirusExpression system by GibcoBRL. For example, ROC1 was cloned into thepFastBacHtb vector with a N-terminal His6 (SEQ ID NO:21) tag, while CUL1was inserted into the pFastBac1 vector without any fusing tag. Aftertransposition and Bacmid DNA transfection into SF-9 cells, Baculoviruseswere harvested, amplified, and used to co-infect Hi-S cells for proteinexpression.

Materials and Methods

Cells are harvested, washed once with cold PBS, and resuspended in about6 volumes of lysis buffer (20 mM Tris, 20% glycerol, 0.5 M Nacl, 15 mMimidazole, 1 mM TCEP plus Complete -EDTA Free Protease inhibitortablets, 1 tablet/25 ml of resuspended cells, pH 8.0.). The suspensionis then sonicated 3×30 sec, followed by addition of NP40 to a finalconcentration of 0.5% and incubation for 30 min at 4° C. The lysate isthen centrifuged and the supernatant is incubated with pre-equilibrated(lysis buffer+NP40) Ni-NTA Agarose beads (Qiagen, cat # 1000632) for 1to 2 hrs. The pelleted beads are washed 2 times with lysis buffer,resuspended in 1 to 2 volumes of lysis buffer and transferred to adisposable column for elution. Elution is accomplished using 5×1-bedvolume aliquots of Lysis buffer+250 mM imidazole. Elution fractions areanalyzed by SDS-PAGE and appropriate fractions are pooled. The elutionpool is then desalted using either a desalting column or a centrifugalconcentration device (more often used for large volumes.) When usingcentrifugal devices, the eluted pool is diluted 1:1 with lysis bufferthat has no imidazole and spun at the appropriate speed until the volumeis reduced by half. At this point an equal volume of fresh buffer isadded and the device is respun. This is done a total of four timesresulting in a 32 fold exchange.

Example 2 Ubiquitin Conjugation Assay

Ubiquitin conjugating activity of E1 +E2 was measured using thefollowing protocol with FLAG (DYKDDDDK; SEQ ID NO:16)-ubiquitin,purified from E. coli, and the E2 Ubch5c, purified as His-Ubch5c from E.coli.

Materials and Methods

The following procedures were used for assays measuring ubiquitinconjugation. The wells of Nickel-substrate 96-well plates (PierceChemical) are blocked with 100 μl of 1% casein/phosphate buffered saline(PBS) for 1 hour at room temperature, then washed with 200 μl of PBST(0.1% Tween-20 in PBS)3 times. To each well is added the followingFLAG-ubiquitin (SEQ ID NO:14) (see above) reaction solution:

Final Concentration

62.5 mM Tris pH 7.5

6.25 mM MgCl₂

0.75 mM DTT

2.5 mM ATP

2.5 mM NaF

12.5 nM Okadaic acid

100 ng FLAG-ubiquitin (SEQ ID NO:14) (made as described above).

The buffer solution is brought to a final volume of 80 μl withMillipore-filtered water, followed by the addition of 10 μl of DMSO.

To the above solution is then added 10 μl of E1,His-E2 in 20 mM Trisbuffer, pH 7.5, and 5% glycerol. His-E2 is made as described above. E1is obtained commercially (Affiniti Research Products, Exeter, U.K.). Thefollowing amounts of each enzyme are used for these assays: 5 ng/well ofE1; 25 nl/well E2. The reaction is then allowed to proceed at roomtemperature for 1 hour.

Following the ubiquitin conjugation reaction, the wells are washed with200 μl of PBST3 times. For measurement of the enzyme-bound ubiquitin,100 μl of Mouse anti-FLAG (DYKDDDDK; SEQ ID NO:16) (1:10,000) andanti-Mouse Ig-HRP (1:15,000) in PBST are added to each well and allowedto incubate at room temperature for 1 hour. The wells are then washedwith 200 μl of PBST 3 times, followed by the addition of 100 μl ofluminol substrate (⅕ dilution). Luminescence for each well is thenmeasured using a fluorimeter.

Results

Ubiquitin Activating and Conjugating Activity

FIG. 1A shows the luminescence measured for E1 alone and for E1+his-E2,as described above.

Example 3 Ubiquitin Ligase Assay

Ubiquitin ligase activity of E1 +E2+E3 was measured using the followingprotocol with FLAG-ubiquitin (SEQ ID NO:14), purified from E. coli, theE2 UbchSc, purified as GST-UbchSc from E. coli with the GST tag removed,and the E3 His-ROC/Cul1 complex purified from Hi-5 cells by Baculovirusco-infection. This assay was also used to show the effects of candidatemodulators on ubiquitin ligase activity.

Materials and Methods

The wells of Nickel-substrate 96-well plates (Pierce Chemical) areblocked with 100 μl of 1% casein/phosphate buffered saline (PBS) for 1hour at room temperature, then washed with 200 μl of PBST (0.1% Tween-20in PBS) 3 times. To each well is added the following FLAG-ubiquitin (SEQID NO:14) (see above) reaction solution:

Final Concentration

62.5 mM Tris pH7.5

6.25 mM MgCl2

0.75 mM DTT

2.5 mM ATP

2.5 mM NaF

12.5 nM Okadaic acid

100 ng FLAG-ubiquitin (SEQ ID NO:14) (made as described above).

The buffer solution is brought to a final volume of 80 μl withMillipore-filtered water.

For assays directed to identifying modulators of ubiquitin ligaseactivity, 10 μl of a candidate modulator compound in DMSO is then addedto the solution. If no candidate modulator is added, 10 μl of DMSO isadded to the solution.

To the above solution is then added 10 μl of ubiquitination enzymes in20 mM Tris buffer, pH 7.5, and 5% glycerol. F2-Ubch5c andE3-HisROC1/Cul1 are made as described above. E1 is obtained commercially(Affiniti Research Products, Exeter, U.K.). The following amounts ofeach enzyme are used for these assays: 5 ng/well of E1; 25 nl/well E2;and 100 ng/well His-E3. The reaction is then allowed to proceed at roomtemperature for 1 hour.

Following the ubiquitination reaction, the wells are washed with 200 μlof PBST 3 times. For measurement of the enzyme-bound ubiquitin, 100 μlof Mouse anti-FLAG (DYKDDDDK; SEQ ID NO:16) (1:10,000) and anti-Mouse IgHRP (1:15,000) in PBST are added to each well and allowed to incubate atroom temperature for 1 hour. The wells are then washed with 200 μl ofPBST 3 times, followed by the addition of 100 μl of luminol substrate (⅕dilution). Luminescence for each well is then measured using afluorimeter.

Results

Ubiquitin Ligase Activity

FIG. 1 shows the luminescence measured for several differentcombinations of ubiquitination enzymes. In these experiments, only E3was in the form His-E3. The luminescence measurements show that theassay specifically measures the activity of the entire ubiquitinationenzyme cascade, which requires the presence of all three ubiquitinationenzymes in the reaction.

Variations of Composition Components

FIG. 2A shows the relative effect of varying the amount of E1 onubiquitin ligase activity in the above procedure, in presence andabsence of DMSO. The addition of about 10 ng per 100 μl reactionsolution provides maximum ubiquitin ligase activity with the othercomponents of the composition kept as detailed above. The presence ofDMSO does not significantly affect the activity of the ubiquitinationenzymes.

The relative effect of varying E3 and ubiquitin concentration of thereaction composition is shown in FIG. 2B. Generally speaking, maximumubiquitin ligase activity was obtained with 200 to 300 ng per 100 μl ofE3 at each concentration of ubiquitin, while increasing ubiquitinconcentration generally increased ubiquitin ligase activity at eachconcentration of E3.

It was also found that blocking of the wells with 1% casein improved thesignal to noise ratio over either no blocking or blocking with 5% bovineserum albumen (BSA). Background was determined after combining all ofthe components as above except His-E3 and measuring the resultingfluorescence after pre-treating the wells with 5% BSA, 1% casein ornothing. Results are shown in FIG. 3.

Identification of Modulators of Ubiquitin Ligase Activity

To show that the assay is useful for identifying modulators of ubiquitinligase activity, several candidate modulators were combined at varyingconcentrations with the assay components as described above. FIG. 4shows the results from two identified modulators of ubiquitin ligaseactivity. The modulators decreased ubiquitin ligase activity in adose-dependent fashion for ubiquitination enzyme compositions comprisingeither ROC1/Cul1 or ROC2/Cul5 as the E3 component.

Comparison of the effect of ubiquitin ligase activity modulators onreaction compositions, as described above, either containing E1, E2 andHis-E3 or containing E1, His-E2 and lacking E3 shows whether themodulator affects E3 or an enzyme other than E3. In FIG. 5A, theidentified modulator decreases ubiquitin ligase activity in the presenceof E3, but does not alter activity in the absence of E3, showing thatthe modulator has a specific effect on E3 ligase activity. In contrast,results shown in FIG. 5B for another modulator reveals that thiscompound reduces activity whether or not E3 is present, showing thatthis modulator affects a member of the ubiquitination enzyme cascadeother than E3.

Example 4 FRET Analysis of Ligated Ubiquitin

Ubiquitin was prepared, labeled with either EDANS or fluorescein, andthe fluorescence of each of these labels and their interaction as a FRETpair was measured to show binding of the labeled ubiquitin to E3 andFRET activity in the bound ubiquitin.

Materials and Methods

Ubiquitin were produced incorporating Cys residues into theFLAG-ubiquitin (SEQ ID NO:14) sequence by site-directed mutagenesisusing either the primer5 ′-CCCCCCAAGCTTTGCATGCAGATTTTCGTGAAGACCCTGACC-3 ′(SEQ ID NO:19)to produce Flag-Cys-ubiquitin (SEQ ID NO:15), or the primer5 ′-CCCCCCAAGCTTGCGTGCATGCAGATTTTCGTGAAGACCCTGACC-3 ′(SEQ ID NO:20)to produce FLAG-Ala-Cys-ubiquitin (SEQ ID NO:18). Protein was expressedand purified as described above.

Either fluoresceine 5-maleimide (peak emission at 515 nm) or1,5-iodacetamide EDANS (IAEDANS; peak emission at 490 nm) was reactedwith the thiol group on the cysteine of the ubiquitin produced as aboveto form a thioether. The labeling was performed in PBS with 1 mM TCEP.Labeled protein was separated from free label by gel filtration.

Ubiquitin ligase assay was performed substantially as described above,with a few modifications no nickel substrate was used in the reactionwells, so all of the components were free in solution. Equal amounts offluoresceine labeled ubiquitin and IAEDANS labeled ubiquitin were used.The reaction was performed at room temperature for 2 hours in a volumeof 100–150 μl, then stopped with 50 μl of 0.5 M EDTA, pH 8.

Following the reaction, the products were separated in PBS with 1 mMTCEP by HPLC on a Superdex-75 HR 10/30 size-exclusion column usingfluorescence emission detection. A larger molecular weight cutoffgel-filtration column (e.g., Superdex 200 HR 10/30) could be used toresolve individual ligation species.

RESULTS

Fluoresceine labeled ubiquitin and IAEDANS labeled ubiquitin was boundto E3 in approximately equal amounts. A comparison of the spectralanalysis of fluorescent emission from the free (unligated) ubiquitinlabeled with both fluorophores and the E3-bound ubiquitin shows adistinct increase in ratio of emission at 515 nm versus 490 nm (FIG.17). This shows that in ligated ubiquitin, the fluorphores on differentubiquitin molecules are sufficiently close for FRET to be measured.

1. A method of identifying a ubiquitination modulator comprising: a)combining, under conditions that favor ubiquitination activity: i)tag1-ubiquitin; ii) a candidate modulator; iii) ubiquitin activatingenzyme (E1); and iv) tag2-ubiquitin conjugating enzyme (E2); b)measuring the amount of tag1-ubiquitin bound to said tag2-ubiquitinconjugating enzyme (E2), whereby a difference in bound ubiquitin ascompared with a reaction performed in the absence of the candidatemodulator indicates that the candidate is a ubiquitination modulator. 2.The method of claim 1, wherein ubiquitin conjugating enzyme (E2) isselected from the group consisting of Ubiquitin conjugating enzyme 5(Ubc5), Ubiquitin conjugating enzyme 3 (Ubc3), Ubiquitin conjugatingenzyme 4 (Ubc4), and Ubiquitin conjugating enzyme X (UbcX).
 3. Themethod of claim 1, wherein tag1 is a label or a partner of a bindingpair.
 4. The method of claim 3, wherein said tag1 label is a fluorescentlabel.
 5. The method of claim 4, wherein said measuring is by measuringluminescence.
 6. The method of claim 3, wherein said tag1 partner of abinding pair is selected from the group consisting of an antigen,biotin, and calmodulin binding protein (CBP).
 7. The method of claim 6,wherein said tag1 partner of a binding pair is labeled by indirectlabeling.
 8. The method of claim 6, wherein said antigen is FLAG(DYKDDDDK; SEQ ID NO:16).
 9. The method of claim 7, wherein saidindirect labeling is with a fluorescent label or a label enzyme.
 10. Themethod of claim 7, wherein said tag1 partner of a binding pair is FLAG(DYKDDDDK; SEQ ID NO:16).
 11. The method of claim 9, wherein saidmeasuring is by measuring luminescence.
 12. Them method of claim 9,wherein said label enzyme is selected from the group consisting ofhorseradish peroxidase, alkaline phosphatase and glucose oxidase. 13.The method of claim 9, wherein said tag1 partner of a binding pair isFLAG and said indirect labeling is via anti-FLAG (DYKDDDDK; SEQ IDNO:16).
 14. The method of claim 12, wherein said label enzyme is reactedwith a label enzyme substrate which produces a fluorescent product. 15.The method of claim 12, wherein said tag1 is FLAG (DYKDDDDK; SEQ IDNO:16).
 16. The method of claim 14, wherein said measuring is bymeasuring luminescence.
 17. The method of claim 3, wherein said tag2 isa surface substrate binding molecule.
 18. The method of claim 17,wherein said surface substrate binding molecule is selected from thegroup consisting of polyhistidine (His-tag) andglutathione-S-transferase (GST)-tag.
 19. The method of claim 18, whereinsaid surface substrate binding molecule is polyhistidine (His-tag). 20.The method of claim 19, wherein said combining and measuring isperformed in a multi-well plate comprising a surface substratecomprising nickel.